Neprilysin inhibitors

ABSTRACT

In one aspect, the invention relates to compounds having the formula XII: 
                         
where R a , R b , R 2 , R 7 , and X are as defined in the specification, or a pharmaceutically acceptable salt thereof. The compounds described herein are prodrugs of compounds having neprilysin inhibition activity. In another aspect, the invention relates to pharmaceutical compositions comprising these compounds; methods of using these compounds; and processes and intermediates for preparing these compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/911,819, filed on Jun. 6, 2013; which application claims the benefitof U.S. Provisional Application No. 61/657,229, filed on Jun. 8, 2012and U.S. Provisional Application No. 61/773,969, filed on Mar. 7, 2013;the entire disclosures of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to novel compounds which are metabolizedin vivo to compounds having activity as neprilysin inhibitors. Theinvention also relates to pharmaceutical compositions comprising thesecompounds, processes and intermediates for preparing these compounds andmethods of using these compounds to treat diseases such as hypertension,heart failure, pulmonary hypertension, and renal disease.

State of the Art

Commonly-assigned U.S. Patent Publication No. 2012/0157383, filed onDec. 14, 2011 to Gendron et al., describes novel compounds that haveactivity as neprilysin inhibitors, the disclosure of which isincorporated herein by reference. In particular, compounds of the genus:

are described. Depending upon the variables, compounds within this genuscan be referred to as being in the active form or as being a prodrug,which is metabolized in vivo to generate the active form of thecompound.

In spite of these compounds however, there remains a need for compoundsand prodrugs within this genus that have different metabolic andcleavage properties. For example, there remains a need for activecompounds and/or prodrug compounds having improved oral absorption andfor prodrug compounds that undergo rapid cleavage to form the activecompound. This invention is directed to that need.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a compound of formula XII:

where:

(i) R^(a) is H; R^(b) is Cl; X is

and

R² is H, R⁴ is —OH, and R⁷ is selected from —CH₂CF₃, —(CH₂)₂CF₃,—CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃,—(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —(CH₂)₂OCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

or R² is H, R⁴ is selected from —O-benzyl, —OCHR^(c)OC(O)—C₁₋₄alkyl,—OCH₂OC(O)CHR^(d)—NH₂, and —OCH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and R⁷is selected from H and —CH₂OC(O)CH₃; or R² is selected from—C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂, —C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and—P(O)(OR^(e))₂, R⁴ is —OH, and R⁷ is H; or

(ii) R^(a) is H; R^(b) is Cl; X is

and

R² is H, R³ is —OH and R⁷ is selected from —CH₂CF₃, —(CH₂)₂CF₃,—CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃,—(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —(CH₂)₂OCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

or R³ is selected from —OC(O)CH₂CH₃, —OC(O)CH₂CH(CH₃)₂, —OC(O)-phenyl,—OCH₂OC(O)CHR^(d)—NH₂, and —OCH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and R⁷is H; or R² is selected from —C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂,—C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and —P(O)(OR^(e))₂, R³ is —OH, and R⁷ isH; or

(iii) R^(a) is H; R^(b) is Cl; X is

and

R² is H, R³ is —OH and R⁷ is selected from —CH₂CH₃, —CH₂CH(CH₃)₂,—CH₂CF₃, —(CH₂)₂CF₃, —CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂,—CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃, —(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃,—(CH₂)₂OCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl, —CHR^(c)OC(O)O—C₂₋₄alkyl,—CHR^(c)OC(O)O-cyclohexyl, —C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

or R² is H, R³ is selected from —OC(O)CH₂CH₃, —OC(O)CH₂CH(CH₃)₂,—OC(O)-phenyl, —OCH₂OC(O)CHR^(d)—NH₂, and—OCH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and R⁷ is H; or R² is selected from—C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂, —C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and—P(O)(OR^(e))₂, R³ is —OH, and R⁷ is H; or

(iv) R^(a) is F; R^(b) is Cl; X is

and

R² is H and R⁷ is selected from H, —CH₂CH₃, —CH₂CF₃, —(CH₂)₂CF₃,—CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃,—(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —(CH₂)₂OCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

or R² is selected from —C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂,—C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and —P(O)(OR^(e))₂, and R⁷ is H; or

(v) R^(a) is H; R^(b) is Cl; X is

and

R² is H and R⁷ is selected from H, —CH₂CH₃, —CH₂CF₃, —(CH₂)₂CF₃,—CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃,—(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —(CH₂)₂OCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

or R² is selected from —C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂,—C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and —P(O)(OR^(e))₂, and R⁷ is H; or

(vi) R^(a) is H; R^(b) is Cl; X is

and

R² is H and R⁷ is selected from H, —CH₂CH₃, —CH₂CF₃, —(CH₂)₂CF₃,—CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃,—(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —(CH₂)₂OCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

or R² is selected from —C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂,—C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and —P(O)(OR^(e))₂, and R⁷ is H; or

(vii) R^(a) is H; R^(b) is Cl; X is

R is H or —CH₃; and

R² is H and R⁷ is selected from —CH₂CH₃, —CH₂CF₃, —(CH₂)₂CF₃,—CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃,—(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —(CH₂)₂OCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

or R² is selected from —C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂,—C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and —P(O)(OR^(e))₂, and R⁷ is H; or

(viii) R^(a) is F; R^(b) is Cl; X is

and

R² is H, R⁴ is —OH, and R⁷ is selected from —CH₂CF₃, —(CH₂)₂CF₃,—CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃,—(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —(CH₂)₂OCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

or R² is H, R⁴ is selected from —O-benzyl, —OCHR^(c)OC(O)—C₁₋₄alkyl,—OCH₂OC(O)CH[CH(CH₃)₂]NH₂, —OCH₂OC(O)CH[CH(CH₃)₂]—NHC(O)OCH₃ and

and R⁷ is H; or R² is selected from —C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂,—C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and —P(O)(OR^(e))₂, R⁴ is —OH, and R⁷ isH; or

(ix) R^(a) is H; R^(b) is Cl; X is

and

R² is H and R⁷ is selected from —CH₂CH₃, —CH₂CF₃, —(CH₂)₂CF₃,—CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃,—(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —(CH₂)₂OCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

or R² is selected from —C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂,—C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and —P(O)(OR^(e))₂, and R⁷ is H; or

(x) R^(a) is H; R^(b) is H; X is

and

R² and R⁴ are H, and R⁷ is selected from —CH₂CF₃, —(CH₂)₂CF₃,—CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃,—(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and benzyl; or R² is H, R⁴ isselected from —CH₂OC(O)CH[CH(CH₃)₂]—NHC(O)OCH₃ and—CH₂OC(O)CH[CH(CH₃)₂]NH₂, and R⁷ is H; or R² is selected from—C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂, —C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and—P(O)(OR^(e))₂, R⁴ is H, and R⁷ is H; or R² is H, R⁴ is—CH₂OP(O)(OR^(e))₂ or —CH₂OC(O)CH[CH(CH₃)₂]NH₂, and R⁷ is —CH₂CH₃; or R²is —C(O)CH[CH(CH₃)₂]NH₂, R⁴ is H, and R⁷ is —CH₂CH₃; or

(xi) R^(a) is H; R^(b) is Cl; X is

and

R² and R⁴ are H, and R⁷ is selected from H, —CH₂CH₃, —CH₂CF₃,—(CH₂)₂CF₃, —CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃,—CH(CH₃)CF₂CF₃, —(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —(CH₂)₂OCH₃,—CHR^(c)OC(O)—C₁₋₄alkyl, —CHR^(c)OC(O)O—C₂₋₄alkyl,—CHR^(c)OC(O)O-cyclohexyl, —C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and benzyl; or R² is H, R⁴ isselected from —CH₂OC(O)CH[CH(CH₃)₂]—NHC(O)OCH₃ and—CH₂OC(O)CH[CH(CH₃)₂]NH₂, and R⁷ is H; or R² is selected from—C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂, —C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and—P(O)(OR^(e))₂, R⁴ is H, and R⁷ is H; or R² is H, R⁴ is—CH₂OP(O)(OR^(e))₂ or —CH₂OC(O)CH[CH(CH₃)₂]NH₂, and R⁷ is —CH₂CH₃; or R²is —C(O)CH[CH(CH₃)₂]NH₂, R⁴ is H, and R⁷ is —CH₂CH₃;

where each R^(c) is independently H or —C₁₋₃alkyl; each R^(d) isindependently H, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) isindependently H, —C₁₋₆alkyl, or phenyl;

or a pharmaceutically acceptable salt thereof.

The present invention provides compounds which are metabolized in vivoto compounds that have been found to possess neprilysin (NEP) enzymeinhibition activity. Accordingly, compounds of the invention areexpected to be useful and advantageous as therapeutic agents fortreating patients suffering from a disease or disorder that is treatedby inhibiting the NEP enzyme or by increasing the levels of its peptidesubstrates. Thus, one aspect of the invention relates to a method oftreating hypertension, heart failure, or renal disease, comprisingadministering to a patient a therapeutically effective amount of acompound of the invention.

Another aspect of the invention relates to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a compound of theinvention.

Yet another aspect of the invention relates to processes andintermediates useful for preparing compounds of the invention. Anotheraspect of the invention relates to a process of preparing apharmaceutically acceptable salt of a compound of formula I, comprisingcontacting a compound of formula I in free acid or base form with apharmaceutically acceptable base or acid. In other aspects, theinvention relates to products prepared by any of the processes describedherein, as well as novel intermediates used in such process.

Yet another aspect of the invention relates to the use of a compound offormula I or a pharmaceutically acceptable salt thereof, for themanufacture of a medicament, especially for the manufacture of amedicament useful for treating hypertension, heart failure, or renaldisease. Another aspect of the invention relates to use of a compound ofthe invention for inhibiting a NEP enzyme in a mammal. Still anotheraspect of the invention relates to the use of a compound of theinvention as a research tool. Other aspects and embodiments of theinvention are disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

When describing the compounds, compositions, methods and processes ofthe invention, the following terms have the following meanings unlessotherwise indicated.

Additionally, as used herein, the singular forms “a,” “an,” and “the”include the corresponding plural forms unless the context of use clearlydictates otherwise. The terms “comprising”, “including,” and “having”are intended to be inclusive and mean that there may be additionalelements other than the listed elements. All numbers expressingquantities of ingredients, properties such as molecular weight, reactionconditions, and so forth used herein are to be understood as beingmodified in all instances by the term “about,” unless otherwiseindicated. Accordingly, the numbers set forth herein are approximationsthat may vary depending upon the desired properties sought to beobtained by the present invention. At least, and not as an attempt tolimit the application of the doctrine of equivalents to the scope of theclaims, each number should at least be construed in light of thereported significant digits and by applying ordinary roundingtechniques.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms and include, for example,—C₁₋₆alkyl, meaning an alkyl group having from 1 to 6 carbon atoms wherethe carbon atoms are in any acceptable configuration. Representativealkyl groups include, by way of example, methyl, ethyl, n-propyl,isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, n-pentyl, n-hexyl, andthe like.

As used herein, the phrase “having the formula” or “having thestructure” is not intended to be limiting and is used in the same waythat the term “comprising” is commonly used. For example, if onestructure is depicted, it is understood that all stereoisomer andtautomer forms are encompassed, unless stated otherwise.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise unacceptable when used in the invention. Forexample, the term “pharmaceutically acceptable carrier” refers to amaterial that can be incorporated into a composition and administered toa patient without causing unacceptable biological effects or interactingin an unacceptable manner with other components of the composition. Suchpharmaceutically acceptable materials typically have met the requiredstandards of toxicological and manufacturing testing, and include thosematerials identified as suitable inactive ingredients by the U.S. Foodand Drug administration.

The term “pharmaceutically acceptable salt” means a salt prepared from abase or an acid which is acceptable for administration to a patient,such as a mammal (for example, salts having acceptable mammalian safetyfor a given dosage regime). However, it is understood that the saltscovered by the invention are not required to be pharmaceuticallyacceptable salts, such as salts of intermediate compounds that are notintended for administration to a patient. Pharmaceutically acceptablesalts can be derived from pharmaceutically acceptable inorganic ororganic bases and from pharmaceutically acceptable inorganic or organicacids. In addition, when a compound of formula I contains both a basicmoiety, such as an amine, pyridine or imidazole, and an acidic moietysuch as a carboxylic acid or tetrazole, zwitterions may be formed andare included within the term “salt” as used herein. Salts derived frompharmaceutically acceptable inorganic bases include ammonium, calcium,copper, ferric, ferrous, lithium, magnesium, manganic, manganous,potassium, sodium, and zinc salts, and the like. Salts derived frompharmaceutically acceptable organic bases include salts of primary,secondary and tertiary amines, including substituted amines, cyclicamines, naturally-occurring amines and the like, such as arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. Salts derived frompharmaceutically acceptable inorganic acids include salts of boric,carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric orhydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Saltsderived from pharmaceutically acceptable organic acids include salts ofaliphatic hydroxyl acids (for example, citric, gluconic, glycolic,lactic, lactobionic, malic, and tartaric acids), aliphaticmonocarboxylic acids (for example, acetic, butyric, formic, propionicand trifluoroacetic acids), amino acids (for example, aspartic andglutamic acids), aromatic carboxylic acids (for example, benzoic,p-chlorobenzoic, diphenylacetic, gentisic, hippuric, and triphenylaceticacids), aromatic hydroxyl acids (for example, o-hydroxybenzoic,p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylic and3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic acids(for example, fumaric, maleic, oxalic and succinic acids), glucoronic,mandelic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids(for example, benzenesulfonic, camphosulfonic, edisylic, ethanesulfonic,isethionic, methanesulfonic, naphthalenesulfonic,naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic andp-toluenesulfonic acids), xinafoic acid, and the like.

As used herein, the term “prodrug” is intended to mean an inactive (orsignificantly less active) precursor of a drug that is converted intoits active form in the body under physiological conditions, for example,by normal metabolic processes. Such compounds may not necessarilypossess pharmacological activity at NEP, but may be administered orallyor parenterally and thereafter metabolized in the body to form acompound that is pharmacologically active at NEP.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need thereof, thatis, the amount of drug needed to obtain the desired therapeutic effect.For example, a therapeutically effective amount for treatinghypertension is an amount of compound needed to, for example, reduce,suppress, eliminate, or prevent the symptoms of hypertension, or totreat the underlying cause of hypertension. In one embodiment, atherapeutically effective amount is that amount of drug needed to reduceblood pressure or the amount of drug needed to maintain normal bloodpressure. On the other hand, the term “effective amount” means an amountsufficient to obtain a desired result, which may not necessarily be atherapeutic result. For example, when studying a system comprising a NEPenzyme, an “effective amount” may be the amount needed to inhibit theenzyme.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as hypertension) in apatient, such as a mammal (particularly a human) that includes one ormore of the following: (a) preventing the disease or medical conditionfrom occurring, i.e., preventing the reoccurrence of the disease ormedical condition or prophylactic treatment of a patient that ispre-disposed to the disease or medical condition; (b) ameliorating thedisease or medical condition, i.e., eliminating or causing regression ofthe disease or medical condition in a patient; (c) suppressing thedisease or medical condition, i.e., slowing or arresting the developmentof the disease or medical condition in a patient; or (d) alleviating thesymptoms of the disease or medical condition in a patient. For example,the term “treating hypertension” would include preventing hypertensionfrom occurring, ameliorating hypertension, suppressing hypertension, andalleviating the symptoms of hypertension (for example, lowering bloodpressure). The term “patient” is intended to include those mammals, suchas humans, that are in need of treatment or disease prevention or thatare presently being treated for disease prevention or treatment of aspecific disease or medical condition, as well as test subjects in whichthe crystalline compound is being evaluated or being used in an assay,for example an animal model.

All other terms used herein are intended to have their ordinary meaningas understood by those of ordinary skill in the art to which theypertain.

The compound of the invention contain one or more chiral centers andtherefore, these compounds may be prepared and used in variousstereoisomeric forms. In some embodiments, in order to optimize thetherapeutic activity of the compounds of the invention, e.g., to treathypertension, it may be desirable that the carbon atoms have aparticular (R,R), (S,S), (S,R), or (R,S) configuration or are enrichedin a stereoisomeric form having such configuration. In otherembodiments, the compounds of the invention are present as racemicmixtures. Accordingly, the invention also relates to racemic mixtures,pure stereoisomers (e.g., enantiomers and diastereoisomers),stereoisomer-enriched mixtures, and the like unless otherwise indicated.When a chemical structure is depicted herein without anystereochemistry, it is understood that all possible stereoisomers areencompassed by such structure. Similarly, when a particular stereoisomeris shown or named herein, it will be understood by those skilled in theart that minor amounts of other stereoisomers may be present in thecompositions of the invention unless otherwise indicated, provided thatthe utility of the composition as a whole is not eliminated by thepresence of such other isomers. Individual stereoisomers may be obtainedby numerous methods that are well known in the art, including chiralchromatography using a suitable chiral stationary phase or support, orby chemically converting them into diastereoisomers, separating thediastereoisomers by conventional means such as chromatography orrecrystallization, then regenerating the original stereoisomer.

Additionally, where applicable, all cis-trans or E/Z isomers (geometricisomers), tautomeric forms and topoisomeric forms of the compounds ofthe invention are included within the scope of the invention unlessotherwise specified.

The compounds of the invention, as well as those compounds used in theirsynthesis, may also include isotopically-labeled compounds, that is,where one or more atoms have been enriched with atoms having an atomicmass different from the atomic mass predominately found in nature.Examples of isotopes that may be incorporated into the compounds offormula I, for example, include, but are not limited to, ²H, ³H, ¹³C,¹⁴C, ¹⁵N, ¹⁸, ¹⁷O, ³⁵S, ³⁶Cl, and ¹⁸F. Of particular interest arecompounds of formula I enriched in tritium or carbon-14 which can beused, for example, in tissue distribution studies; compounds of theinvention enriched in deuterium especially at a site of metabolismresulting, for example, in compounds having greater metabolic stability;and compounds of formula I enriched in a positron emitting isotope, suchas ¹¹C, ¹⁸F, ¹⁵O and ¹³N, which can be used, for example, in PositronEmission Topography (PET) studies.

The nomenclature used herein to name the compounds of the invention isillustrated in the Examples herein. This nomenclature has been derivedusing the commercially available AutoNom software (MDL, San Leandro,Calif.).

U.S. Patent Publication No. 2012/0157383 specifically disclosed(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicacid, which is represented by formula I′:

In one embodiment, this compound is referred to as the active form andis administered as a prodrug, which is metabolized in vivo to form thecompound of formula I′. U.S. Patent Publication No. 2012/0157383 alsodisclosed the ethyl ester prodrug of the compound of formula I′.

One aspect of the invention relates to other prodrugs of the compound offormula I′. These prodrugs are represented by formula XII, where R^(a)is H, R^(b) is Cl, and X is:

In one embodiment, these compounds are represented by formula I:

where R² is H, R⁴ is —OH, and R⁷ is selected from —CH₂CF₃, —(CH₂)₂CF₃,—CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃,—(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —(CH₂)₂OCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

or R² is H, R⁴ is selected from —O-benzyl, —OCHR^(c)OC(O)—C₁₋₄alkyl,—OCH₂OC(O)CHR^(d)—NH₂, and —OCH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and R⁷is selected from H and —CH₂OC(O)CH₃; or R² is selected from—C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂, —C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and—P(O)(OR^(e))₂, R⁴ is —OH, and R⁷ is H; where each R^(c) isindependently H or —C₁₋₃alkyl; each R^(d) is independently H, —CH₃,—CH(CH₃)₂, phenyl, or benzyl; and each R^(e) is independently H,—C₁₋₆alkyl, or phenyl; or a pharmaceutically acceptable salt thereof.

In one particular embodiment of the compounds of Formula I, R² is H, R⁴is —OH and R⁷ is selected from —CH₂CF₃, —(CH₂)₂CF₃, —CH₂CF₂CF₃,—CH₂OC(O)CH₃, —CH₂OC(O)(CH₂)₂CH₃, —CH₂OC(O)CH[CH(CH₃)₂]NH₂,—CH₂OC(O)CH[CH(CH₃)₂]—NHC(O)OCH₃, benzyl, and

or R² is H, R⁴ is —OCH₂OC(O)CH₃ and R⁷ is selected from H and—CH₂OC(O)CH₃; or R² is H, R⁴ is selected from —OCH₂OC(O)(CH₂)₂CH₃,—CH₂OC(O)CH[CH(CH₃)₂]NH₂, and —OCH₂OC(O)CH[CH(CH₃)₂]—NHC(O)OCH₃, and R⁷is H; or R² is H, R⁴ is —O-benzyl and R⁷ is H.

The compound(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-hydroxy-1-methyl-1H-pyrazole-3-carbonyl)amino]pentanoicacid is also specifically disclosed in U.S. Patent Publication No.2012/0157383, and is represented by formula II′:

In one embodiment, this compound is referred to as the active form andis administered as a prodrug, which is metabolized in vivo to form thecompound of formula II′. U.S. Patent Publication No. 2012/0157383 alsodisclosed the isobutyl ester prodrug of the compound of formula II′.

Another aspect of the invention relates to other prodrugs of thecompound of formula II′. These prodrugs are represented by formula XII,where R^(a) is H, R^(b) is Cl, and X is:

In one embodiment, these compounds are represented by formula II:

where R² is H, R³ is —OH and R⁷ is selected from —CH₂CF₃, —(CH₂)₂CF₃,—CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃,—(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —(CH₂)₂OCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

or R³ is selected from —OC(O)CH₂CH₃, —OC(O)CH₂CH(CH₃)₂, —OC(O)-phenyl,—OCH₂OC(O)CHR^(d)—NH₂, and —OCH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and R⁷is H; or R² is selected from —C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂,—C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and —P(O)(OR^(e))₂, R³ is —OH, and R⁷ isH; where each R^(c) is independently H or —C₁₋₃alkyl; each R^(d) isindependently H, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) isindependently H, —C₁₋₆alkyl, or phenyl; or a pharmaceutically acceptablesalt thereof.

In one particular embodiment of the compounds of Formula II, R² is H, R³is —OH and R⁷ is selected from —CH₂CH₃, —CH₂CF₂CF₃, —CH(CH₂CH₃)CF₃,—CH(CH₃)CF₂CF₃, —CH₂OC(O)(CH₂)₂CH₃, —CH₂OC(O)CH[CH(CH₃)₂]NH₂, benzyl,and

The compound(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-hydroxy-1-pyridin-2-yl-1H-pyrazole-3-carbonyl)amino]pentanoicacid is also specifically disclosed in U.S. Patent Publication No.2012/0157383, and is represented by formula III′:

In one embodiment, this compound is referred to as the active form andis administered as a prodrug, which is metabolized in vivo to form thecompound of formula III′.

Another aspect of the invention relates to other prodrugs of thecompound of formula III′. These prodrugs are represented by formula XII,where R^(a) is H, R^(b) is Cl, and X is:

In one embodiment, these compounds are represented by formula III:

where R² is H, R³ is —OH and R⁷ is selected from —CH₂CH₃, —CH₂CH(CH₃)₂,—CH₂CF₃, —(CH₂)₂CF₃, —CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂,—CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃, —(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃,—(CH₂)₂OCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl, —CHR^(c)OC(O)O—C₂₋₄alkyl,—CHR^(c)OC(O)O-cyclohexyl, —C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

or R² is H, R³ is selected from —OC(O)CH₂CH₃, —OC(O)CH₂CH(CH₃)₂,—OC(O)-phenyl, —OCH₂OC(O)CHR^(d)—NH₂, and—OCH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and R⁷ is H; or R² is selected from—C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂, —C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and—P(O)(OR^(e))₂, R³ is —OH, and R⁷ is H; where each R^(c) isindependently H or —C₁₋₃alkyl; each R^(d) is independently H, —CH₃,—CH(CH₃)₂, phenyl, or benzyl; and each R^(e) is independently H,—C₁₋₆alkyl, or phenyl; or a pharmaceutically acceptable salt thereof.

In one particular embodiment of the compounds of Formula III, R² is H,R³ is —OH and R⁷ is selected from —CH₂CH₃, —CH₂CH(CH₃)₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

where R^(d) is —CH(CH₃)₂. In another embodiment of the compounds ofFormula III, R² is H, R³ is —OCH₂OC(O)CH[CH(CH₃)₂]NH₂, and R⁷ is H. Instill another embodiment of the compounds of Formula III, R² is—C(O)CH[CH(CH₃)₂]NH₂, R³ is —OH, and R⁷ is H.

Another aspect of the invention relates to a compound of formula XII,where R^(a) is F, R^(b) is Cl, and X is:

In one embodiment, these compounds are represented by formula IV:

where R² is H and R⁷ is selected from H, —CH₂CH₃, —CH₂CF₃, —(CH₂)₂CF₃,—CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃,—(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —(CH₂)₂OCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

or R² is selected from —C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂,—C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and —P(O)(OR^(e))₂, and R⁷ is H; whereeach R^(c) is independently H or —C₁₋₃alkyl; each R^(d) is independentlyH, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) is independentlyH, —C₁₋₆alkyl, or phenyl; or a pharmaceutically acceptable salt thereof.

In one particular embodiment of the compounds of Formula IV, R² is H andR⁷ is selected from H and —CH₂CH₃.

Another aspect of the invention relates to a compound of formula XII,where R^(a) is H, R^(b) is Cl, and X is:

In one embodiment, these compounds are represented by formula V:

where R² is H and R⁷ is selected from H, —CH₂CH₃, —CH₂CF₃, —(CH₂)₂CF₃,—CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃,—(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —(CH₂)₂OCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

or R² is selected from —C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂,—C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and —P(O)(OR^(e))₂, and R⁷ is H; whereeach R^(c) is independently H or —C₁₋₃alkyl; each R^(d) is independentlyH, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) is independentlyH, —C₁₋₆alkyl, or phenyl; or a pharmaceutically acceptable salt thereof.

In one particular embodiment of the compounds of Formula V, R² is H andR⁷ is selected from H and —CH₂CH₃.

Another aspect of the invention relates to a compound of formula XII,where R^(a) is H, R^(b) is Cl, and X is:

In one embodiment, these compounds are represented by formula VI:

where R² is H and R⁷ is selected from H, —CH₂CH₃, —CH₂CF₃, —(CH₂)₂CF₃,—CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃,—(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —(CH₂)₂OCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

or R² is selected from —C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂,—C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and —P(O)(OR^(e))₂, and R⁷ is H; whereeach R^(c) is independently H or —C₁₋₃alkyl; each R^(d) is independentlyH, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) is independentlyH, —C₁₋₆alkyl, or phenyl; or a pharmaceutically acceptable salt thereof.

In one particular embodiment of the compounds of Formula VI, R² is H andR⁷ is selected from H, —CH₂OC(O)CH₃, —CH₂OC(O)OCH₂CH₃,—CH₂OC(O)OCH(CH₃)₂, and —C(O)CH[CH(CH₃)₂]—NHC(O)OCH₃.

The compound(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-methoxyisoxazole-5-carbonyl)amino]pentanoicacid is also specifically disclosed in U.S. Patent Publication No.2012/0157383, and is represented by formula VII′:

In one embodiment, this compound is referred to as the active form andis administered as a prodrug, which is metabolized in vivo to form thecompound of formula VII′.

Another aspect of the invention relates to other prodrugs of thecompound of formula VII′. These prodrugs are represented by formula XII,where R^(a) is H, R^(b) is Cl, and X is:

where R is H or —CH₃. In one embodiment, these compounds are representedby formula VII:

where R² is H and R⁷ is selected from —CH₂CH₃, —CH₂CF₃, —(CH₂)₂CF₃,—CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃,—(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —(CH₂)₂OCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

or R² is selected from —C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂,—C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and —P(O)(OR^(e))₂, and R⁷ is H; whereeach R^(c) is independently H or —C₁₋₃alkyl; each R^(d) is independentlyH, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) is independentlyH, —C₁₋₆alkyl, or phenyl; or a pharmaceutically acceptable salt thereof.

In one particular embodiment of the compounds of Formula VII, R is —CH₃,R² is H, and R⁷ is selected from —CH₂OC(O)CH₃, —CH₂OC(O)OCH(CH₃)₂,—CH₂OC(O)OCH₂CH₃, and —CH₂OC(O)CH[CH(CH₃)₂]—NHC(O)OCH₃.

The compound(2R,4R)-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-[(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicacid is also specifically disclosed in U.S. Patent Publication No.2012/0157383, and is represented by formula VIII′:

In one embodiment, this compound is referred to as the active form andis administered as a prodrug, which is metabolized in vivo to form thecompound of formula VIII′. U.S. Patent Publication No. 2012/0157383 alsodisclosed the isopropyl ester, ethyl ester, isobutyl ester, and heptylester prodrugs of the compound of formula VIII′.

Another aspect of the invention relates to other prodrugs of thecompound of formula VIII′. These prodrugs are represented by formulaXII, where R^(a) is F, R^(b) is Cl, and X is:

In one embodiment, these compounds are represented by formula VIIIa orVIIIb:

where R² is H, R⁴ is —OH, and R⁷ is selected from —CH₂CF₃, —(CH₂)₂CF₃,—CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃,—(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —(CH₂)₂OCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

or R² is H, R⁴ is selected from —O-benzyl, —OCHR^(c)OC(O)—C₁₋₄alkyl,—OCH₂OC(O)CH[CH(CH₃)₂]NH₂, —OCH₂OC(O)CH[CH(CH₃)₂]—NHC(O)OCH₃ and

and R⁷ is H; or R² is selected from —C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂,—C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and —P(O)(OR^(e))₂, R⁴ is —OH, and R⁷ isH; where each R^(c) is independently H or —C₁₋₃alkyl; each R^(d) isindependently H, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) isindependently H, —C₁₋₆alkyl, or phenyl; or a pharmaceutically acceptablesalt thereof.

In one particular embodiment of the compounds of Formula VIIIa andVIIIb, R² is H, R⁴ is —OH and R⁷ is selected from —CH₂CF₂CF₃,—CH₂OC(O)CH₃, —CH₂OC(O)(CH₂)₂CH₃, —CH₂OC(O)OCH₂CH₃, —CH₂OC(O)OCH(CH₃)₂,—CH(CH₃)OC(O)O-cyclohexyl, —CH₂OC(O)CH[CH(CH₃)₂]—NHC(O)OCH₃, and

or R² is H, R⁴ is selected from —OCH₂OC(O)(CH₂)₂CH₃,—OCH₂OC(O)CH[CH(CH₃)₂]NH₂, —OCH₂OC(O)CH[CH(CH₃)₂]—NHC(O)OCH₃, and

and R⁷ is H.

The compound(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-oxo-1-phenyl-4,5-dihydro-1H-[1,2,4]triazole-3-carbonyl)amino]pentanoicacid is also specifically disclosed in U.S. Patent Publication No.2012/0157383, and is represented by formula IX′:

In one embodiment, this compound is referred to as the active form andis administered as a prodrug, which is metabolized in vivo to form thecompound of formula IX′.

Another aspect of the invention relates to other prodrugs of thecompound of formula IX′. These prodrugs are represented by formula XII,where R^(a) is H, R^(b) is Cl, and X is:

In one embodiment, these compounds are represented by formula IX:

where R² is H and R⁷ is selected from —CH₂CH₃, —CH₂CF₃, —(CH₂)₂CF₃,—CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃,—(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —(CH₂)₂OCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, benzyl, and

or R² is selected from —C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂,—C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and —P(O)(OR^(e))₂, and R⁷ is H; whereeach R^(c) is independently H or —C₁₋₃alkyl; each R^(d) is independentlyH, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) is independentlyH, —C₁₋₆alkyl, or phenyl; or a pharmaceutically acceptable salt thereof.

In one particular embodiment of the compounds of Formula IX, R² is H andR⁷ is selected from —CH₂OC(O)OCH₂CH₃ and—CH₂OC(O)CH[CH(CH₃)₂]—NHC(O)OCH₃.

The compound(2R,4R)-4-[(5-acetyl-2H-pyrazole-3-carbonyl)amino]-5-biphenyl-4-yl-2-hydroxypentanoicacid is also specifically disclosed in U.S. Patent Publication No.2012/0157383, and is represented by formula X′:

In one embodiment, this compound is referred to as the active form andis administered as a prodrug, which is metabolized in vivo to form thecompound of formula X′. U.S. Patent Publication No. 2012/0157383 alsodisclosed the ethyl ester, butyl ester, methoxyethyl ester, medoxomilester, mofetil ester, and methanesulfonylethyl ester prodrugs of thecompound of formula X′.

Another aspect of the invention relates to other prodrugs of thecompound of formula X′. These prodrugs are represented by formula XII,where R^(a) is H, R^(b) is H, and X is:

In one embodiment, these compounds are represented by formula Xa or Xb:

where R² and R⁴ are H, and R⁷ is selected from —CH₂CF₃, —(CH₂)₂CF₃,—CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃,—(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and benzyl; or R² is H, R⁴ isselected from —CH₂OC(O)CH[CH(CH₃)₂]—NHC(O)OCH₃ and—CH₂OC(O)CH[CH(CH₃)₂]NH₂, and R⁷ is H; or R² is selected from—C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂, —C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and—P(O)(OR^(e))₂, R⁴ is H, and R⁷ is H; or R² is H, R⁴ is—CH₂OP(O)(OR^(e))₂ or —CH₂OC(O)CH[CH(CH₃)₂]NH₂, and R⁷ is —CH₂CH₃; or R²is —C(O)CH[CH(CH₃)₂]NH₂, R⁴ is H, and R⁷ is —CH₂CH₃; and where eachR^(e) is independently H or —C₁₋₃alkyl; each R^(d) is independently H,—CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) is independently H,—C₁₋₆alkyl, or phenyl; or a pharmaceutically acceptable salt thereof.

In one particular embodiment of the compounds of Formula Xa and Xb, R²is H, R⁴ is —CH₂—OP(O)(OH)₂ or —CH₂OC(O)CH[CH(CH₃)₂]NH₂, and R⁷ is—CH₂CH₃. In another embodiment of the compounds of Formula Xa and Xb, R²is —C(O)CH[CH(CH₃)₂]NH₂, R⁴ is H, and R⁷ is —CH₂CH₃.

Another aspect of the invention relates to a compound of formula XII,where R^(a) is H, R^(b) is Cl, and X is:

In one embodiment, these compounds are represented by formula XIa orXIb:

where R² and R⁴ are H, and R⁷ is selected from H, —CH₂CH₃, —CH₂CF₃,—(CH₂)₂CF₃, —CH₂CF₂CH₃, —CH₂CF₂CF₃, —C(CH₃)(CF₃)₂, —CH(CH₂CH₃)CF₃,—CH(CH₃)CF₂CF₃, —(CH₂)₂₋₃OH, —CH₂CH(NH₂)COOCH₃, —(CH₂)₂OCH₃,—CHR^(c)OC(O)—C₁₋₄alkyl, —CHR^(c)OC(O)O—C₂₋₄alkyl,—CHR^(c)OC(O)O-cyclohexyl, —C₂₋₄alkylene-N(CH₃)₂, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and benzyl; or R² is H, R⁴ isselected from —CH₂OC(O)CH[CH(CH₃)₂]—NHC(O)OCH₃ and—CH₂OC(O)CH[CH(CH₃)₂]NH₂, and R⁷ is H; or R² is selected from—C(O)—C₁₋₆alkyl, —C(O)CHR^(d)—NH₂, —C(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, and—P(O)(OR^(e))₂, R⁴ is H, and R⁷ is H; or R² is H, R⁴ is—CH₂OP(O)(OR^(e))₂ or —CH₂OC(O)CH[CH(CH₃)₂]NH₂, and R⁷ is —CH₂CH₃; or R²is —C(O)CH[CH(CH₃)₂]NH₂, R⁴ is H, and R⁷ is —CH₂CH₃; and where eachR^(c) is independently H or —C₁₋₃alkyl; each R^(d) is independently H,—CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) is independently H,—C₁₋₆alkyl, or phenyl; or a pharmaceutically acceptable salt thereof.

In one particular embodiment of the compounds of Formula XIa and XIb,R², R⁴, and R⁷ are H. In another embodiment of the compounds of FormulaXIa and XIb, R² and R⁴ are H, and R⁷ is —CH₂OC(O)OCH₂CH₃.

General Synthetic Procedures

Compounds of the invention can be prepared from readily availablestarting materials using the following general methods, the proceduresset forth in the Examples, or by using other methods, reagents, andstarting materials that are known to those of ordinary skill in the art.Although the following procedures may illustrate a particular embodimentof the invention, it is understood that other embodiments of theinvention can be similarly prepared using the same or similar methods orby using other methods, reagents and starting materials known to thoseof ordinary skill in the art. It will also be appreciated that wheretypical or preferred process conditions (for example, reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. In some instances, reactions were conducted at roomtemperature and no actual temperature measurement was taken. It isunderstood that room temperature can be taken to mean a temperaturewithin the range commonly associated with the ambient temperature in alaboratory environment, and will typically be in the range of about 18°C. to about 30° C. In other instances, reactions were conducted at roomtemperature and the temperature was actually measured and recorded.While optimum reaction conditions will typically vary depending onvarious reaction parameters such as the particular reactants, solventsand quantities used, those of ordinary skill in the art can readilydetermine suitable reaction conditions using routine optimizationprocedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions and reagents for protection anddeprotection of such functional groups are well-known in the art.Protecting groups other than those illustrated in the proceduresdescribed herein may be used, if desired. For example, numerousprotecting groups, and their introduction and removal, are described inT. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis,Fourth Edition, Wiley, New York, 2006, and references cited therein.

Carboxy-protecting groups are suitable for preventing undesiredreactions at a carboxy group, and examples include, but are not limitedto, methyl, ethyl, t-butyl, benzyl (Bn), p-methoxybenzyl (PMB),9-fluorenylmethyl (Fm), trimethylsilyl (TMS), t-butyldimethylsilyl(TBDMS), diphenylmethyl (benzhydryl, DPM) and the like. Amino-protectinggroups are suitable for preventing undesired reactions at an aminogroup, and examples include, but are not limited to, t-butoxycarbonyl(BOC), trityl (Tr), benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl(Fmoc), formyl, trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS), andthe like.

Standard deprotection techniques and reagents are used to remove theprotecting groups, and may vary depending upon which group is used. Forexample, sodium or lithium hydroxide is commonly used when thecarboxy-protecting group is methyl, an acid such as TFA or HCl (e.g.,4.0 M HCl in 1,4-dioxane) is commonly used when the carboxy-protectinggroup is ethyl or t-butyl, and H₂/Pd/C may be used when thecarboxy-protecting group is benzyl. A BOC amino-protecting group can beremoved using an acidic reagent such as TFA in DCM or HCl in1,4-dioxane, while a Cbz amino-protecting group can be removed byemploying catalytic hydrogenation conditions such as H₂ (1 atm) and 10%Pd/C in an alcoholic solvent (“H₂/Pd/C”).

Leaving groups are functional groups or atoms that can be displaced byanother functional group or atom in a substitution reaction, such as anucleophilic substitution reaction. By way of example, representativeleaving groups include chloro, bromo and iodo groups; sulfonic estergroups, such as mesylate, tosylate, brosylate, nosylate and the like;and acyloxy groups, such as acetoxy, trifluoroacetoxy and the like.

Suitable bases for use in these schemes include, by way of illustrationand not limitation, potassium carbonate, calcium carbonate, sodiumcarbonate, triethylamine (Et₃N), pyridine,1,8-diazabicyclo-[5.4.0]undec-7-ene (DBU), N,N-diisopropylethylamine(DIPEA), 4-methylmorpholine, sodium hydroxide, potassium hydroxide,potassium t-butoxide, and metal hydrides.

Suitable inert diluents or solvents for use in these schemes include, byway of illustration and not limitation, tetrahydrofuran (THF),acetonitrile (MeCN), N,N-dimethylformamide (DMF), N,N-dimethylacetamide(DMA), dimethyl sulfoxide (DMSO), toluene, dichloromethane (DCM),chloroform (CHCl₃), carbon tetrachloride (CCl₄), 1,4-dioxane, methanol,ethanol, water, diethyl ether, acetone, and the like.

Suitable carboxylic acid/amine coupling reagents includebenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (HATU), 1,3-dicyclohexylcarbodiimide (DCC),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC),carbonyldiimidazole (CDI), 1-hydroxybenzotriazole (HOBt), and the like.Coupling reactions are conducted in an inert diluent in the presence ofa base such as DIPEA, and are performed under conventional amidebond-forming conditions.

All reactions are typically conducted at a temperature within the rangeof about −78 C to 100° C., for example at room temperature. Reactionsmay be monitored by use of thin layer chromatography (TLC), highperformance liquid chromatography (HPLC), and/or LCMS until completion.Reactions may be complete in minutes, or may take hours, typically from1-2 hours and up to 48 hours. Upon completion, the resulting mixture orreaction product may be further treated in order to obtain the desiredproduct. For example, the resulting mixture or reaction product may besubjected to one or more of the following procedures: concentrating orpartitioning (for example, between EtOAc and water or between 5% THF inEtOAc and 1M phosphoric acid); extraction (for example, with EtOAc,CHCl₃, DCM, chloroform); washing (for example, with saturated aqueousNaCl, saturated aqueous NaHCO₃, Na₂CO₃ (5%), CHCl₃ or 1M NaOH); drying(for example, over MgSO₄, over Na₂SO₄, or in vacuo); filtering;crystallizing (for example, from EtOAc and hexanes); being concentrated(for example, in vacuo); and/or purification (e.g., silica gelchromatography, flash chromatography, preparative HPLC, reversephase-HPLC, or crystallization).

By way of illustration, compounds of the invention, as well as theirsalts, can be prepared as shown in Schemes I-IV.

Scheme I is a transesterification reaction. Generally, this reactioninvolves reacting the ester with heat, the desired alcohol (HO—R⁷) and asuitable acid catalyst, for example hydrochloric acid. The HO—R⁷alcohols are either commercially available or can be prepared bytechniques that are known in the art or described herein. ExemplaryHO—R⁷ compounds include HO—CH₂CF₃, HO—(CH₂)₂CF₃, HO—CH₂CF₂CH₃,HO—CH₂CF₂CF₃, HO—C(CH₃)(CF₃)₂, HO—CH(CH₂CH₃)CF₃, HO—CH(CH₃)CF₂CF₃,benzyl alcohol, and

Scheme II is a nucleophilic substitution reaction, where L is a suitableleaving group. Generally, this reaction is conducted in the presence ofa suitable base such as triethylamine in a suitable inert diluent orsolvent such as acetone. The L-R⁷ compounds are either commerciallyavailable or can be prepared by techniques that are known in the art ordescribed herein. Exemplary L-R⁷ compounds include Br—(CH₂)₂OH,Br—(CH₂)₃OH, Br—(CH₂)₂OCH₃, Br—CH₂OC(O)CH₃, Cl—CH₂OC(O)(CH₂)₂CH₃,Cl—CH₂OC(O)OCH₂CH₃, Cl—CH₂OC(O)OCH(CH₃)₂, Cl—CH₂OC(O)O-cyclohexyl,(S)-2-benzyloxycarbonylamino-3-methyl-butyric acid chloromethyl ester,and (S)-2-t-butoxycarbonylamino-3-methyl-butyric acid chloromethylester.

Alternately, in Scheme II, an alcohol have be used in place of the L-R⁷,for example HO—C₂₋₄alkylene-N(CH₃)₂ in a coupling reaction using HOBtand EDC.

Scheme III is a nucleophilic substitution reaction, where L is asuitable leaving group. Generally, this reaction is conducted in thepresence of a suitable base such as N,N-diisopropylethylamine in asuitable inert diluent or solvent such as dichloromethane. The L-R²compound is either commercially available or can be prepared bytechniques that are known in the art or described herein. Exemplary L-R²compounds include Cl—C(O)—CH₃, Cl—C(O)—CH(CH₃)₂, andCl—C(O)—CH₂CH(CH₃)₂.

Scheme IV is a coupling reaction, where P is H or a suitableamino-protecting group. When P is an amino protecting group, the processfurther comprises deprotecting the compound before or in situ with thecoupling step. Exemplary coupling reagents include HATU and HOBt withEDC. Generally, these reactions are conducted in the presence of a basesuch as DIPEA or 4-methylmorpholine, and an inert diluent or solventssuch as DMF or DMA. The carboxylic acid starting materials are generallycommercially available or can be prepared using procedures that areknown in the art.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of the invention orintermediates thereof are described in the Examples set forth below.

Utility

The compounds of formula I′-III′ and VII′-X′ have activity as neprilysininhibitors, and are expected to have therapeutic utility as neprilysininhibitors. Prodrugs of these compounds, once metabolized in vivo, areexpected to have the same utility. Thus, when discussing the activity ofthe compounds of the invention, it is understood that these prodrugshave the expected activity once metabolized.

Exemplary assays include by way of illustration and not limitation,assays that measure NEP inhibition. Useful secondary assays includeassays to measure ACE inhibition and aminopeptidase P (APP) inhibition(e.g., as described in Sulpizio et al. (2005) JPET 315:1306-1313). Apharmacodynamic assay to assess the in vivo inhibitory potencies for ACEand NEP in anesthetized rats is described in Seymour et al. (1985)Hypertension 7(Suppl I):I-35-I-42 and Wigle et al. (1992) Can. J.Physiol. Pharmacol. 70:1525-1528), where ACE inhibition is measured asthe percent inhibition of the angiotensin I pressor response and NEPinhibition is measured as increased urinary cyclic guanosine3′,5′-monophosphate (cGMP) output.

There are also many in vivo assays that can be used. The consciousspontaneously hypertensive rat (SHR) model is a renin dependenthypertension model. See for example, Intengan et al. (1999) Circulation100(22):2267-2275 and Badyal et al. (2003) Indian Journal ofPharmacology 35:349-362. The conscious desoxycorticosterone acetate-salt(DOCA-salt) rat model is a volume dependent hypertension model that isuseful for measuring NEP activity. See for example, Trapani et al.(1989) J. Cardiovasc. Pharmacol. 14:419-424, Intengan et al. (1999)Hypertension 34(4):907-913, and Badyal et al. (2003) supra). TheDOCA-salt model is particularly useful for evaluating the ability of atest compound to reduce blood pressure as well as to measure a testcompound's ability to prevent or delay a rise in blood pressure. TheDahl salt-sensive (DSS) hypertensive rat model is a model ofhypertension that is sensitive to dietary salt (NaCl), and is described,for example, in Rapp (1982) Hypertension 4:753-763. The ratmonocrotaline model of pulmonary arterial hypertension described, forexample, in Kato et al. (2008) J. Cardiovasc. Pharmacol. 51(1):18-23, isa reliable predictor of clinical efficacy for the treatment of pulmonaryarterial hypertension. Heart failure animal models include the DSS ratmodel for heart failure and the aorto-caval fistula model (AV shunt),the latter of which is described, for example, in Norling et al. (1996)J. Amer. Soc. Nephrol. 7:1038-1044. Other animal models, such as the hotplate, tail-flick and formalin tests, can be used to measure theanalgesic properties of a compound, as well as the spinal nerve ligation(SNL) model of neuropathic pain. See, for example, Malmberg et al.(1999) Current Protocols in Neuroscience 8.9.1-8.9.15. Other propertiesand utilities of the compounds can be demonstrated using various invitro and in vivo assays well known to those skilled in the art.

The compounds of the invention are expected to be useful for thetreatment and/or prevention of medical conditions responsive to NEPinhibition. Thus it is expected that patients suffering from a diseaseor disorder that is treated by inhibiting the NEP enzyme or byincreasing the levels of its peptide substrates, can be treated byadministering a therapeutically effective amount of a compound of theinvention. For example, by inhibiting NEP, the compound is expected topotentiate the biological effects of endogenous peptides that aremetabolized by NEP, such as the natriuretic peptides, bombesin,bradykinins, calcitonin, endothelins, enkephalins, neurotensin,substance P and vasoactive intestinal peptide. Thus, the compounds areexpected to have other physiological actions, for example, on the renal,central nervous, reproductive and gastrointestinal systems.

Cardiovascular Diseases

By potentiating the effects of vasoactive peptides like the natriureticpeptides and bradykinin, compounds of the invention are expected to findutility in treating and/or preventing medical conditions such ascardiovascular diseases. See, for example, Roques et al. (1993)Pharmacol. Rev. 45:87-146 and Dempsey et al. (2009) Amer. J. ofPathology 174(3):782-796. Cardiovascular diseases of particular interestinclude hypertension and heart failure. Hypertension includes, by way ofillustration and not limitation: primary hypertension, which is alsoreferred to as essential hypertension or idiopathic hypertension;secondary hypertension; hypertension with accompanying renal disease;severe hypertension with or without accompanying renal disease;pulmonary hypertension, including pulmonary arterial hypertension; andresistant hypertension. Heart failure includes, by way of illustrationand not limitation: congestive heart failure; acute heart failure;chronic heart failure, for example with reduced left ventricularejection fraction (also referred to as systolic heart failure) or withpreserved left ventricular ejection fraction (also referred to asdiastolic heart failure); and acute and chronic decompensated heartfailure, with or without accompanying renal disease. Thus, oneembodiment of the invention relates to a method for treatinghypertension, particularly primary hypertension or pulmonary arterialhypertension, comprising administering to a patient a therapeuticallyeffective amount of a compound of the invention.

For treatment of primary hypertension, the therapeutically effectiveamount is typically the amount that is sufficient to lower the patient'sblood pressure. This would include both mild-to-moderate hypertensionand severe hypertension. When used to treat hypertension, the compoundmay be administered in combination with other therapeutic agents such asaldosterone antagonists, angiotensin-converting enzyme inhibitors anddual-acting angiotensin-converting enzyme/neprilysin inhibitors,angiotensin-converting enzyme 2 (ACE2) activators and stimulators,angiotensin-II vaccines, anti-diabetic agents, anti-lipid agents,anti-thrombotic agents, AT₁ receptor antagonists and dual-acting AT₁receptor antagonist/neprilysin inhibitors, β₁-adrenergic receptorantagonists, dual-acting β-adrenergic receptor antagonist/α₁-receptorantagonists, calcium channel blockers, diuretics, endothelin receptorantagonists, endothelin converting enzyme inhibitors, neprilysininhibitors, natriuretic peptides and their analogs, natriuretic peptideclearance receptor antagonists, nitric oxide donors, non-steroidalanti-inflammatory agents, phosphodiesterase inhibitors (specificallyPDE-V inhibitors), prostaglandin receptor agonists, renin inhibitors,soluble guanylate cyclase stimulators and activators, and combinationsthereof. In one particular embodiment of the invention, a compound ofthe invention is combined with an AT₁ receptor antagonist, a diuretic, acalcium channel blocker, or a combination thereof, and used to treatprimary hypertension. In another particular embodiment of the invention,a compound of the invention is combined with an AT₁ receptor antagonist,and used to treat hypertension with accompanying renal disease.

For treatment of pulmonary arterial hypertension, the therapeuticallyeffective amount is typically the amount that is sufficient to lower thepulmonary vascular resistance. Other goals of therapy are to improve apatient's exercise capacity. For example, in a clinical setting, thetherapeutically effective amount can be the amount that improves apatient's ability to walk comfortably for a period of 6 minutes(covering a distance of approximately 20-40 meters). When used to treatpulmonary arterial hypertension the compound may be administered incombination with other therapeutic agents such as α-adrenergicantagonists, β₁-adrenergic receptor antagonists, β₂-adrenergic receptoragonists, angiotensin-converting enzyme inhibitors, anticoagulants,calcium channel blockers, diuretics, endothelin receptor antagonists,PDE-V inhibitors, prostaglandin analogs, selective serotonin reuptakeinhibitors, and combinations thereof. In one particular embodiment ofthe invention, a compound of the invention is combined with a PDE-Vinhibitor or a selective serotonin reuptake inhibitor and used to treatpulmonary arterial hypertension.

Another embodiment of the invention relates to a method for treatingheart failure, in particular congestive heart failure (including bothsystolic and diastolic congestive heart failure), comprisingadministering to a patient a therapeutically effective amount of acompound of the invention. Typically, the therapeutically effectiveamount is the amount that is sufficient to lower blood pressure and/orimprove renal functions. In a clinical setting, the therapeuticallyeffective amount can be the amount that is sufficient to improve cardiachemodynamics, like for instance reduction in wedge pressure, rightatrial pressure, filling pressure, and vascular resistance. In oneembodiment, the compound is administered as an intravenous dosage form.When used to treat heart failure, the compound may be administered incombination with other therapeutic agents such as adenosine receptorantagonists, advanced glycation end product breakers, aldosteroneantagonists, AT₁ receptor antagonists, β-adrenergic receptorantagonists, dual-acting β-adrenergic receptor antagonist/α₁-receptorantagonists, chymase inhibitors, digoxin, diuretics, endothelinconverting enzyme (ECE) inhibitors, endothelin receptor antagonists,natriuretic peptides and their analogs, natriuretic peptide clearancereceptor antagonists, nitric oxide donors, prostaglandin analogs, PDE-Vinhibitors, soluble guanylate cyclase activators and stimulators, andvasopressin receptor antagonists. In one particular embodiment of theinvention, a compound of the invention is combined with an aldosteroneantagonist, a β₁-adrenergic receptor antagonist, an AT₁ receptorantagonist, or a diuretic, and used to treat congestive heart failure.

Diarrhea

As NEP inhibitors, compounds of the invention are expected to inhibitthe degradation of endogenous enkephalins and thus such compounds mayalso find utility for the treatment of diarrhea, including infectiousand secretory/watery diarrhea. See, for example, Baumer et al. (1992)Gut 33:753-758; Farthing (2006) Digestive Diseases 24:47-58; andMarcais-Collado (1987) Eur. J. Pharmacol. 144(2):125-132. When used totreat diarrhea, compounds of the invention may be combined with one ormore additional antidiarrheal treatments.

Renal Diseases

By potentiating the effects of vasoactive peptides like the natriureticpeptides and bradykinin, compounds of the invention are expected toenhance renal function (see Chen et al. (1999) Circulation100:2443-2448; Lipkin et al. (1997) Kidney Int. 52:792-801; and Dussauleet al. (1993) Clin. Sci. 84:31-39) and find utility in treating and/orpreventing renal diseases. Renal diseases of particular interest includediabetic nephropathy, chronic kidney disease, proteinuria, andparticularly acute kidney injury or acute renal failure (see Sharkovskaet al. (2011) Clin. Lab. 57:507-515 and Newaz et al. (2010) RenalFailure 32:384-390). When used to treat renal disease, the compound maybe administered in combination with other therapeutic agents such asangiotensin-converting enzyme inhibitors, AT₁ receptor antagonists, anddiuretics.

Preventative Therapy

By potentiating the effects of the natriuretic peptides, compounds ofthe invention are also expected to be useful in preventative therapy,due to the antihypertrophic and antifibrotic effects of the natriureticpeptides (see Potter et al. (2009) Handbook of Experimental Pharmacology191:341-366), for example in preventing the progression of cardiacinsufficiency after myocardial infarction, preventing arterialrestenosis after angioplasty, preventing thickening of blood vesselwalls after vascular operations, preventing atherosclerosis, andpreventing diabetic angiopathy.

Glaucoma

By potentiating the effects of the natriuretic peptides, compounds ofthe invention are expected to be useful to treat glaucoma. See, forexample, Diestelhorst et al. (1989) International Ophthalmology12:99-101. When used to treat glaucoma, compounds of the invention maybe combined with one or more additional anti-glaucoma agents.

Pain Relief

As NEP inhibitors, compounds of the invention are expected to inhibitthe degradation of endogenous enkephalins and thus such compounds mayalso find utility as analgesics. See, for example, Roques et al. (1980)Nature 288:286-288 and Thanawala et al. (2008) Current Drug Targets9:887-894. When used to treat pain, compounds of the invention may becombined with one or more additional antinociceptive drugs such asaminopeptidase N or dipeptidyl peptidase III inhibitors, non-steroidalanti-inflammatory agents, monoamine reuptake inhibitors, musclerelaxants, NMDA receptor antagonists, opioid receptor agonists,5-HT_(1D) serotonin receptor agonists, and tricyclic antidepressants.

Other Utilities

Due to their NEP inhibition properties, compounds of the invention arealso expected to be useful as antitussive agents, as well as findutility in the treatment of portal hypertension associated with livercirrhosis (see Sansoe et al. (2005) J. Hepatol. 43:791-798), cancer (seeVesely (2005) J. Investigative Med. 53:360-365), depression (see Nobleet al. (2007) Exp. Opin. Ther. Targets 11:145-159), menstrual disorders,preterm labor, pre-eclampsia, endometriosis, reproductive disorders (forexample, male and female infertility, polycystic ovarian syndrome,implantation failure), and male and female sexual dysfunction, includingmale erectile dysfunction and female sexual arousal disorder. Morespecifically, the compounds of the invention are expected to be usefulin treating female sexual dysfunction (see Pryde et al. (2006) J. Med.Chem. 49:4409-4424), which is often defined as a female patient'sdifficulty or inability to find satisfaction in sexual expression. Thiscovers a variety of diverse female sexual disorders including, by way ofillustration and not limitation, hypoactive sexual desire disorder,sexual arousal disorder, orgasmic disorder and sexual pain disorder.When used to treat such disorders, especially female sexual dysfunction,compounds of the invention may be combined with one or more of thefollowing secondary agents: PDE-V inhibitors, dopamine agonists,estrogen receptor agonists and/or antagonists, androgens, and estrogens.Due to their NEP inhibition properties, compounds of the invention arealso expected to have anti-inflammatory properties, and are expected tohave utility as such, particularly when used in combination withstatins.

Recent studies suggest that NEP plays a role in regulating nervefunction in insulin-deficient diabetes and diet induced obesity. Coppeyet al. (2011) Neuropharmacology 60:259-266. Therefore, due to their NEPinhibition properties, compounds of the invention are also expected tobe useful in providing protection from nerve impairment caused bydiabetes or diet induced obesity.

The amount of the compound of the invention administered per dose or thetotal amount administered per day may be predetermined or it may bedetermined on an individual patient basis by taking into considerationnumerous factors, including the nature and severity of the patient'scondition, the condition being treated, the age, weight, and generalhealth of the patient, the tolerance of the patient to the active agent,the route of administration, pharmacological considerations such as theactivity, efficacy, pharmacokinetics and toxicology profiles of thecompound and any secondary agents being administered, and the like.Treatment of a patient suffering from a disease or medical condition(such as hypertension) can begin with a predetermined dosage or a dosagedetermined by the treating physician, and will continue for a period oftime necessary to prevent, ameliorate, suppress, or alleviate thesymptoms of the disease or medical condition. Patients undergoing suchtreatment will typically be monitored on a routine basis to determinethe effectiveness of therapy. For example, in treating hypertension,blood pressure measurements may be used to determine the effectivenessof treatment. Similar indicators for other diseases and conditionsdescribed herein, are well known and are readily available to thetreating physician. Continuous monitoring by the physician will insurethat the optimal amount of the compound of the invention will beadministered at any given time, as well as facilitating thedetermination of the duration of treatment. This is of particular valuewhen secondary agents are also being administered, as their selection,dosage, and duration of therapy may also require adjustment. In thisway, the treatment regimen and dosing schedule can be adjusted over thecourse of therapy so that the lowest amount of active agent thatexhibits the desired effectiveness is administered and, further, thatadministration is continued only so long as is necessary to successfullytreat the disease or medical condition.

Research Tools

Since the compounds of the invention are metabolized in vivo tocompounds having activity as neprilysin inhibitors, they are also usefulas a research tools for investigating or studying biological systems orsamples having a NEP enzyme, for example to study diseases where the NEPenzyme or its peptide substrates plays a role. Accordingly, one aspectof the invention relates to a method of using a compound of theinvention as a research tool, comprising conducting a biological assayusing a compound of the invention. Any suitable biological system orsample having a NEP enzyme may be employed in such studies which may beconducted either in vitro or in vivo. Representative biological systemsor samples suitable for such studies include, but are not limited to,cells, cellular extracts, plasma membranes, tissue samples, isolatedorgans, mammals (such as mice, rats, guinea pigs, rabbits, dogs, pigs,humans, and so forth), and the like, with mammals being of particularinterest. In one particular embodiment of the invention, NEP enzymeactivity in a mammal is inhibited by administering a NEP-inhibitingamount of a compound of the invention. These compounds can also be usedas research tools by conducting biological assays using such compounds.

When used as a research tool, a biological system or sample comprising aNEP enzyme is typically contacted with a NEP enzyme-inhibiting amount ofa compound of the invention. After the biological system or sample isexposed to the compound, the effects of inhibiting the NEP enzyme aredetermined using conventional procedures and equipment, such as bymeasuring receptor binding in a binding assay or measuringligand-mediated changes in a functional assay. Exposure encompassescontacting cells or tissue with the compound, administering thecrystalline compound to a mammal, for example by i.p., p.o, i.v., s.c.,or inhaled administration, and so forth. This determining step caninvolve measuring a response (a quantitative analysis) or can involvemaking an observation (a qualitative analysis). Measuring a responseinvolves, for example, determining the effects of the compound on thebiological system or sample using conventional procedures and equipment,such as enzyme activity assays and measuring enzyme substrate or productmediated changes in functional assays. The assay results can be used todetermine the activity level as well as the amount of compound necessaryto achieve the desired result, that is, a NEP enzyme-inhibiting amount.Typically, the determining step will involve determining the effects ofinhibiting the NEP enzyme.

Additionally, the compounds of the invention can be used as researchtools for evaluating other chemical compounds, and thus are also usefulin screening assays to discover, for example, new compounds havingNEP-inhibiting activity. Thus another aspect of the invention relates toa method of evaluating a test compound in a biological assay,comprising: (a) conducting a biological assay with a test compound toprovide a first assay value; (b) conducting the biological assay with acompound of the invention to provide a second assay value; wherein step(a) is conducted either before, after or concurrently with step (b); and(c) comparing the first assay value from step (a) with the second assayvalue from step (b). Exemplary biological assays include a NEP enzymeinhibition assay. In this manner, the compounds of the invention areused as standards in an assay to allow comparison of the resultsobtained with a test compound and with the compound of the invention toidentify those test compounds that have about equal or superioractivity, if any. For example, pK_(i) data for a test compound or agroup of test compounds is compared to the pK_(i) data for a compound ofthe invention to identify those test compounds that have the desiredproperties, for example, test compounds having a pK_(i) value aboutequal or superior to the compound of the invention, if any. This aspectof the invention includes, as separate embodiments, both the generationof comparison data (using the appropriate assays) and the analysis oftest data to identify test compounds of interest.

Still another aspect of the invention relates to a method of studying abiological system or sample comprising a NEP enzyme, the methodcomprising: (a) contacting the biological system or sample with acompound of the invention; and (b) determining the effects caused by thecompound on the biological system or sample.

Pharmaceutical Compositions and Formulations

Compounds of the invention are typically administered to a patient inthe form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to, oral,rectal, vaginal, nasal, inhaled, topical (including transdermal),ocular, and parenteral modes of administration. Further, the compoundsof the invention may be administered, for example orally, in multipledoses per day (for example, two, three, or four times daily), in asingle daily dose or a single weekly dose. It will be understood thatany form of the compounds of the invention, (that is, free base, freeacid, pharmaceutically acceptable salt, solvate, etc.) that is suitablefor the particular mode of administration can be used in thepharmaceutical compositions discussed herein.

Accordingly, in one embodiment, the invention relates to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of the invention. The compositions may containother therapeutic and/or formulating agents if desired. When discussingcompositions, the “compound of the invention” may also be referred toherein as the “active agent,” to distinguish it from other components ofthe formulation, such as the carrier. Thus, it is understood that theterm “active agent” includes compounds of formula I as well aspharmaceutically acceptable salts, solvates and prodrugs of thatcompound.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the invention. Thoseskilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,such as in bulk compositions, or less than a therapeutically effectiveamount, that is, individual unit doses designed for multipleadministration to achieve a therapeutically effective amount. Typically,the composition will contain from about 0.01-95 wt % of active agent,including, from about 0.01-30 wt %, such as from about 0.01-10 wt %,with the actual amount depending upon the formulation itself, the routeof administration, the frequency of dosing, and so forth. In oneembodiment, a composition suitable for an oral dosage form, for example,may contain about 5-70 wt %, or from about 10-60 wt % of active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable composition for a particular mode of administration iswell within the scope of those skilled in the pharmaceutical arts.Additionally, carriers or excipients used in such compositions arecommercially available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; compressed propellant gases, such aschlorofluorocarbons and hydrofluorocarbons; and other non-toxiccompatible substances employed in pharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with a pharmaceuticallyacceptable carrier and one or more optional ingredients. The resultinguniformly blended mixture may then be shaped or loaded into tablets,capsules, pills, canisters, cartridges, dispensers and the like usingconventional procedures and equipment.

In one embodiment, the pharmaceutical compositions are suitable for oraladministration. Suitable compositions for oral administration may be inthe form of capsules, tablets, pills, lozenges, cachets, dragees,powders, granules; solutions or suspensions in an aqueous or non-aqueousliquid; oil-in-water or water-in-oil liquid emulsions; elixirs orsyrups; and the like; each containing a predetermined amount of theactive agent.

When intended for oral administration in a solid dosage form (capsules,tablets, pills and the like), the composition will typically comprisethe active agent and one or more pharmaceutically acceptable carriers,such as sodium citrate or dicalcium phosphate. Solid dosage forms mayalso comprise: fillers or extenders, such as starches, microcrystallinecellulose, lactose, sucrose, glucose, mannitol, and/or silicic acid;binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; humectants, such as glycerol;disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; solution retarding agents, such as paraffin; absorptionaccelerators, such as quaternary ammonium compounds; wetting agents,such as cetyl alcohol and/or glycerol monostearate; absorbents, such askaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants may also be presentin the pharmaceutical compositions. Exemplary coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate,carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, and the like. Examples of pharmaceutically acceptableantioxidants include: water-soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfate, sodiumsulfite and the like; oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin,propyl gallate, alpha-tocopherol, and the like; and metal-chelatingagents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions may also be formulated to provide slow or controlledrelease of the active agent using, by way of example, hydroxypropylmethyl cellulose in varying proportions or other polymer matrices,liposomes and/or microspheres. In addition, the pharmaceuticalcompositions of the invention may contain opacifying agents and may beformulated so that they release the active agent only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes. The activeagent can also be in micro-encapsulated form, optionally with one ormore of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (for example, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.Suspensions may contain suspending agents such as, for example,ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminium metahydroxide, bentonite,agar-agar and tragacanth, and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof the invention may be packaged in a unit dosage form. The term “unitdosage form” refers to a physically discrete unit suitable for dosing apatient, that is, each unit containing a predetermined quantity of theactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

In another embodiment, the compositions of the invention are suitablefor inhaled administration, and will typically be in the form of anaerosol or a powder. Such compositions are generally administered usingwell-known delivery devices, such as a nebulizer, dry powder, ormetered-dose inhaler. Nebulizer devices produce a stream of highvelocity air that causes the composition to spray as a mist that iscarried into a patient's respiratory tract. An exemplary nebulizerformulation comprises the active agent dissolved in a carrier to form asolution, or micronized and combined with a carrier to form a suspensionof micronized particles of respirable size. Dry powder inhalersadminister the active agent as a free-flowing powder that is dispersedin a patient's air-stream during inspiration. An exemplary dry powderformulation comprises the active agent dry-blended with an excipientsuch as lactose, starch, mannitol, dextrose, polylactic acid,polylactide-co-glycolide, and combinations thereof. Metered-doseinhalers discharge a measured amount of the active agent usingcompressed propellant gas. An exemplary metered-dose formulationcomprises a solution or suspension of the active agent in a liquefiedpropellant, such as a chlorofluorocarbon or hydrofluoroalkane. Optionalcomponents of such formulations include co-solvents, such as ethanol orpentane, and surfactants, such as sorbitan trioleate, oleic acid,lecithin, glycerin, and sodium lauryl sulfate. Such compositions aretypically prepared by adding chilled or pressurized hydrofluoroalkane toa suitable container containing the active agent, ethanol (if present)and the surfactant (if present). To prepare a suspension, the activeagent is micronized and then combined with the propellant.Alternatively, a suspension formulation can be prepared by spray dryinga coating of surfactant on micronized particles of the active agent. Theformulation is then loaded into an aerosol canister, which forms aportion of the inhaler.

Compounds of the invention can also be administered parenterally (forexample, by subcutaneous, intravenous, intramuscular, or intraperitonealinjection). For such administration, the active agent is provided in asterile solution, suspension, or emulsion. Exemplary solvents forpreparing such formulations include water, saline, low molecular weightalcohols such as propylene glycol, polyethylene glycol, oils, gelatin,fatty acid esters such as ethyl oleate, and the like. Parenteralformulations may also contain one or more anti-oxidants, solubilizers,stabilizers, preservatives, wetting agents, emulsifiers, and dispersingagents. Surfactants, additional stabilizing agents or pH-adjustingagents (acids, bases or buffers) and anti-oxidants are particularlyuseful to provide stability to the formulation, for example, to minimizeor avoid hydrolysis of ester and amide linkages, or dimerization ofthiols that may be present in the compound. These formulations may berendered sterile by use of a sterile injectable medium, a sterilizingagent, filtration, irradiation, or heat. In one particular embodiment,the parenteral formulation comprises an aqueous cyclodextrin solution asthe pharmaceutically acceptable carrier. Suitable cyclodextrins includecyclic molecules containing six or more α-D-glucopyranose units linkedat the 1,4 positions by a linkages as in amylase, β-cyclodextrin orcycloheptaamylose. Exemplary cyclodextrins include cyclodextrinderivatives such as hydroxypropyl and sulfobutyl ether cyclodextrinssuch as hydroxypropyl-β-cyclodextrin and sulfobutyl etherβ-cyclodextrin. Exemplary buffers for such formulations includecarboxylic acid-based buffers such as citrate, lactate and maleatebuffer solutions.

Compounds of the invention can also be administered transdermally usingknown transdermal delivery systems and excipients. For example, thecompound can be admixed with permeation enhancers, such as propyleneglycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and thelike, and incorporated into a patch or similar delivery system.Additional excipients including gelling agents, emulsifiers and buffers,may be used in such transdermal compositions if desired.

Secondary Agents

The compounds of the invention may be useful as the sole treatment of adisease or may be combined with one or more additional therapeuticagents in order to obtain the desired therapeutic effect. Thus, in oneembodiment, pharmaceutical compositions of the invention contain otherdrugs that are co-administered with a compound of the invention. Forexample, the composition may further comprise one or more drugs (alsoreferred to as “secondary agents(s)”). Such therapeutic agents are wellknown in the art, and include adenosine receptor antagonists,α-adrenergic receptor antagonists, β₁-adrenergic receptor antagonists,β₂-adrenergic receptor agonists, dual-acting β-adrenergic receptorantagonist/α₁-receptor antagonists, advanced glycation end productbreakers, aldosterone antagonists, aldosterone synthase inhibitors,aminopeptidase N inhibitors, androgens, angiotensin-converting enzymeinhibitors and dual-acting angiotensin-converting enzyme/neprilysininhibitors, angiotensin-converting enzyme 2 activators and stimulators,angiotensin-II vaccines, anticoagulants, anti-diabetic agents,antidiarrheal agents, anti-glaucoma agents, anti-lipid agents,antinociceptive agents, anti-thrombotic agents, AT₁ receptor antagonistsand dual-acting AT₁ receptor antagonist/neprilysin inhibitors andmultifunctional angiotensin receptor blockers, bradykinin receptorantagonists, calcium channel blockers, chymase inhibitors, digoxin,diuretics, dopamine agonists, endothelin converting enzyme inhibitors,endothelin receptor antagonists, HMG-CoA reductase inhibitors,estrogens, estrogen receptor agonists and/or antagonists, monoaminereuptake inhibitors, muscle relaxants, natriuretic peptides and theiranalogs, natriuretic peptide clearance receptor antagonists, neprilysininhibitors, nitric oxide donors, non-steroidal anti-inflammatory agents,N-methyl d-aspartate receptor antagonists, opioid receptor agonists,phosphodiesterase inhibitors, prostaglandin analogs, prostaglandinreceptor agonists, renin inhibitors, selective serotonin reuptakeinhibitors, sodium channel blocker, soluble guanylate cyclasestimulators and activators, tricyclic antidepressants, vasopressinreceptor antagonists, and combinations thereof. Specific examples ofthese agents are detailed herein.

Accordingly, in yet another aspect of the invention, a pharmaceuticalcomposition comprises a compound of the invention, a second activeagent, and a pharmaceutically acceptable carrier. Third, fourth etc.active agents may also be included in the composition. In combinationtherapy, the amount of compound of the invention that is administered,as well as the amount of secondary agents, may be less than the amounttypically administered in monotherapy.

Compounds of the invention may be physically mixed with the secondactive agent to form a composition containing both agents; or each agentmay be present in separate and distinct compositions which areadministered to the patient simultaneously or at separate times. Forexample, a compound of the invention can be combined with a secondactive agent using conventional procedures and equipment to form acombination of active agents comprising a compound of the invention anda second active agent. Additionally, the active agents may be combinedwith a pharmaceutically acceptable carrier to form a pharmaceuticalcomposition comprising a compound of the invention, a second activeagent and a pharmaceutically acceptable carrier. In this embodiment, thecomponents of the composition are typically mixed or blended to create aphysical mixture. The physical mixture is then administered in atherapeutically effective amount using any of the routes describedherein.

Alternatively, the active agents may remain separate and distinct beforeadministration to the patient. In this embodiment, the agents are notphysically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Suchcompositions can be packaged separately or may be packaged together in akit. When administered at separate times, the secondary agent willtypically be administered less than 24 hours after administration of thecompound of the invention, ranging anywhere from concurrent withadministration of the compound of the invention to about 24 hourspost-dose. This is also referred to as sequential administration. Thus,a compound of the invention can be orally administered simultaneously orsequentially with another active agent using two tablets, with onetablet for each active agent, where sequential may mean beingadministered immediately after administration of the compound of theinvention or at some predetermined time later (for example, one hourlater or three hours later). It is also contemplated that the secondaryagent may be administered more than 24 hours after administration of thecompound of the invention. Alternatively, the combination may beadministered by different routes of administration, that is, one orallyand the other by inhalation.

In one embodiment, the kit comprises a first dosage form comprising acompound of the invention and at least one additional dosage formcomprising one or more of the secondary agents set forth herein, inquantities sufficient to carry out the methods of the invention. Thefirst dosage form and the second (or third, etc.) dosage form togethercomprise a therapeutically effective amount of active agents for thetreatment or prevention of a disease or medical condition in a patient.

Secondary agent(s), when included, are present in a therapeuticallyeffective amount such that they are typically administered in an amountthat produces a therapeutically beneficial effect when co-administeredwith a compound of the invention. The secondary agent can be in the formof a pharmaceutically acceptable salt, solvate, optically purestereoisomer, and so forth. The secondary agent may also be in the formof a prodrug, for example, a compound having a carboxylic acid groupthat has been esterified. Thus, secondary agents listed herein areintended to include all such forms, and are commercially available orcan be prepared using conventional procedures and reagents.

In one embodiment, compounds of the invention are administered incombination with an adenosine receptor antagonist, representativeexamples of which include, but are not limited to, naxifylline,rolofylline, SLV-320, theophylline, and tonapofylline.

In one embodiment, compounds of the invention are administered incombination with an α-adrenergic receptor antagonist, representativeexamples of which include, but are not limited to, doxazosin, prazosin,tamsulosin, and terazosin.

Compounds of the invention may also be administered in combination witha β₁-adrenergic receptor antagonist (“β₁-blockers”). Representativeβ₁-blockers include, but are not limited to, acebutolol, alprenolol,amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol,bisoprolol, bopindolol, bucindolol, bucumolol, bufetolol, bufuralol,bunitrolol, bupranolol, bubridine, butofilolol, carazolol, carteolol,carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol,esmolol, indenolol, labetolol, levobunolol, mepindolol, metipranolol,metoprolol such as metoprolol succinate and metoprolol tartrate,moprolol, nadolol, nadoxolol, nebivalol, nipradilol, oxprenolol,penbutolol, perbutolol, pindolol, practolol, pronethalol, propranolol,sotalol, sufinalol, talindol, tertatolol, tilisolol, timolol,toliprolol, xibenolol, and combinations thereof. In one particularembodiment, the β₁-antagonist is selected from atenolol, bisoprolol,metoprolol, propranolol, sotalol, and combinations thereof. Typically,the β-blocker will be administered in an amount sufficient to providefrom about 2-900 mg per dose.

In one embodiment, compounds of the invention are administered incombination with a β₂-adrenergic receptor agonist, representativeexamples of which include, but are not limited to, albuterol,bitolterol, fenoterol, formoterol, indacaterol, isoetharine,levalbuterol, metaproterenol, pirbuterol, salbutamol, salmefamol,salmeterol, terbutaline, vilanterol, and the like. Typically, theβ₂-adrenergic receptor agonist will be administered in an amountsufficient to provide from about 0.05-500 g per dose.

In one embodiment, compounds of the invention are administered incombination with an advanced glycation end product (AGE) breaker,examples of which include, by way of illustration and not limitation,alagebrium (or ALT-711), and TRC4149.

In another embodiment, compounds of the invention are administered incombination with an aldosterone antagonist, representative examples ofwhich include, but are not limited to, eplerenone, spironolactone, andcombinations thereof. Typically, the aldosterone antagonist will beadministered in an amount sufficient to provide from about 5-300 mg perday.

In one embodiment, compounds of the invention are administered incombination with an aminopeptidase N or dipeptidyl peptidase IIIinhibitor, examples of which include, by way of illustration and notlimitation, bestatin and PC18 (2-amino-4-methylsulfonyl butane thiol,methionine thiol).

Compounds of the invention can also be administered in combination withan angiotensin-converting enzyme (ACE) inhibitor. Representative ACEinhibitors include, but are not limited to, accupril, alacepril,benazepril, benazeprilat, captopril, ceranapril, cilazapril, delapril,enalapril, enalaprilat, fosinopril, fosinoprilat, imidapril, lisinopril,moexipril, monopril, moveltipril, pentopril, perindopril, quinapril,quinaprilat, ramipril, ramiprilat, saralasin acetate, spirapril,temocapril, trandolapril, zofenopril, and combinations thereof.

In a particular embodiment, the ACE inhibitor is selected from:benazepril, captopril, enalapril, lisinopril, ramipril, and combinationsthereof. Typically, the ACE inhibitor will be administered in an amountsufficient to provide from about 1-150 mg per day. In anotherembodiment, compounds of the invention are administered in combinationwith a dual-acting angiotensin-converting enzyme/neprilysin (ACE/NEP)inhibitor, examples of which include, but are not limited to: AVE-0848((4S,7S,12bR)-7-[3-methyl-2(S)-sulfanylbutyramido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]-benzazepine-4-carboxylicacid); AVE-7688 (ilepatril) and its parent compound; BMS-182657(2-[2-oxo-3(S)-[3-phenyl-2(S)-sulfanylpropionamido]-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl]aceticacid); CGS-35601(N-[1-[4-methyl-2(S)-sulfanylpentanamido]cyclopentyl-carbonyl]-L-tryptophan);fasidotril; fasidotrilate; enalaprilat; ER-32935((3R,6S,9aR)-6-[3(S)-methyl-2(S)-sulfanylpentanamido]-5-oxoperhydrothiazolo[3,2-a]azepine-3-carboxylicacid); gempatrilat; MDL-101264((4S,7S,12bR)-7-[2(S)-(2-morpholinoacetylthio)-3-phenylpropionamido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylicacid); MDL-101287([4S-[4α,7α(R*),12bβ]]-7-[2-(carboxymethyl)-3-phenylpropionamido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylicacid); omapatrilat; RB-105(N-[2(S)-(mercaptomethyl)-3(R)-phenylbutyl]-L-alanine); sampatrilat;SA-898((2R,4R)—N-[2-(2-hydroxyphenyl)-3-(3-mercaptopropionyl)thiazolidin-4-ylcarbonyl]-L-phenylalanine);Sch-50690 (N-[1(S)-carboxy-2-[N2-(methanesulfonyl)-L-lysylamino]ethyl]-L-valyl-L-tyrosine);and combinations thereof, may also be included. In one particularembodiment, the ACE/NEP inhibitor is selected from: AVE-7688,enalaprilat, fasidotril, fasidotrilate, omapatrilat, sampatrilat, andcombinations thereof.

In one embodiment, compounds of the invention are administered incombination with an angiotensin-converting enzyme 2 (ACE2) activator orstimulator.

In one embodiment, compounds of the invention are administered incombination with an angiotensin-II vaccine, examples of which include,but are not limited to ATR12181 and CYT006-AngQb.

In one embodiment, compounds of the invention are administered incombination with an anticoagulant, representative examples of whichinclude, but are not limited to: coumarins such as warfarin; heparin;and direct thrombin inhibitors such as argatroban, bivalirudin,dabigatran, and lepirudin.

In yet another embodiment, compounds of the invention are administeredin combination with an anti-diabetic agent. Representative anti-diabeticagents include injectable drugs as well as orally effective drugs, andcombinations thereof. Examples of injectable drugs include, but are notlimited to, insulin and insulin derivatives. Examples of orallyeffective drugs include, but are not limited to: biguanides such asmetformin; glucagon antagonists; α-glucosidase inhibitors such asacarbose and miglitol; dipeptidyl peptidase IV inhibitors (DPP-IVinhibitors) such as alogliptin, denagliptin, linagliptin, saxagliptin,sitagliptin, and vildagliptin; meglitinides such as repaglinide;oxadiazolidinediones; sulfonylureas such as chlorpropamide, glimepiride,glipizide, glyburide, and tolazamide; thiazolidinediones such aspioglitazone and rosiglitazone; and combinations thereof.

In another embodiment, compounds of the invention are administered incombination with antidiarrheal treatments. Representative treatmentoptions include, but are not limited to, oral rehydration solutions(ORS), loperamide, diphenoxylate, and bismuth subsalicylate.

In yet another embodiment, a compound of the invention is administeredin combination with an anti-glaucoma agent. Representative anti-glaucomaagents include, but are not limited to: α-adrenergic agonists such asbrimonidine; β₁-adrenergic receptor antagonists; topical β-blockers suchas betaxolol, levobunolol, and timolol; carbonic anhydrase inhibitorssuch as acetazolamide, brinzolamide, or dorzolamide; cholinergicagonists such as cevimeline and DMXB-anabaseine; epinephrine compounds;miotics such as pilocarpine; and prostaglandin analogs.

In yet another embodiment, compounds of the invention are administeredin combination with an anti-lipid agent. Representative anti-lipidagents include, but are not limited to: cholesteryl ester transferprotein inhibitors (CETPs) such as anacetrapib, dalcetrapib, andtorcetrapib; statins such as atorvastatin, fluvastatin, lovastatin,pravastatin, rosuvastatin and simvastatin; and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with an anti-thrombotic agent. Representativeanti-thrombotic agents include, but are not limited to: aspirin;anti-platelet agents such as clopidogrel, prasugrel, and ticlopidine;heparin, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with an AT₁ receptor antagonist, also known as angiotensinII type 1 receptor blockers (ARBs). Representative ARBs include, but arenot limited to, abitesartan, azilsartan (e.g., azilsartan medoxomil),benzyllosartan, candesartan, candesartan cilexetil, elisartan,embusartan, enoltasosartan, eprosartan, EXP3174, fonsartan, forasartan,glycyllosartan, irbesartan, isoteoline, losartan, medoxomil,milfasartan, olmesartan (e.g., olmesartan medoxomil), opomisartan,pratosartan, ripisartan, saprisartan, saralasin, sarmesin, TAK-591,tasosartan, telmisartan, valsartan, zolasartan, and combinationsthereof. In a particular embodiment, the ARB is selected from azilsartanmedoxomil, candesartan cilexetil, eprosartan, irbesartan, losartan,olmesartan medoxomil, saprisartan, tasosartan, telmisartan, valsartan,and combinations thereof. Exemplary salts and/or prodrugs includecandesartan cilexetil, eprosartan mesylate, losartan potassium salt, andolmesartan medoxomil. Typically, the ARB will be administered in anamount sufficient to provide from about 4-600 mg per dose, withexemplary daily dosages ranging from 20-320 mg per day.

Compounds of the invention may also be administered in combination witha dual-acting agent, such as an AT₁ receptor antagonist/neprilysininhibitor (ARB/NEP), examples of which include, but are not limited to,compounds described in U.S. Publication Nos. 2008/0269305 and2009/0023228, both to Allegretti et al. filed on Apr. 23, 2008, such asthe compound,4′-{2-ethoxy-4-ethyl-5-[((S)-2-mercapto-4-methylpentanoylamino)-methyl]imidazol-1-ylmethyl}-3′-fluorobiphenyl-2-carboxylicacid.

Compounds of the invention may also be administered in combination withmultifunctional angiotensin receptor blockers as described in Kurtz &Klein (2009) Hypertension Research 32:826-834.

In one embodiment, compounds of the invention are administered incombination with a bradykinin receptor antagonist, for example,icatibant (HOE-140). It is expected that this combination therapy maypresent the advantage of preventing angioedema or other unwantedconsequences of elevated bradykinin levels.

In one embodiment, compounds of the invention are administered incombination with a calcium channel blocker. Representative calciumchannel blockers include, but are not limited to, amlodipine, anipamil,aranipine, barnidipine, bencyclane, benidipine, bepridil, clentiazem,cilnidipine, cinnarizine, diltiazem, efonidipine, elgodipine, etafenone,felodipine, fendiline, flunarizine, gallopamil, isradipine, lacidipine,lercanidipine, lidoflazine, lomerizine, manidipine, mibefradil,nicardipine, nifedipine, niguldipine, niludipine, nilvadipine,nimodipine, nisoldipine, nitrendipine, nivaldipine, perhexiline,prenylamine, ryosidine, semotiadil, terodiline, tiapamil, verapamil, andcombinations thereof. In a particular embodiment, the calcium channelblocker is selected from amlodipine, bepridil, diltiazem, felodipine,isradipine, lacidipine, nicardipine, nifedipine, niguldipine,niludipine, nimodipine, nisoldipine, ryosidine, verapamil, andcombinations thereof. Typically, the calcium channel blocker will beadministered in an amount sufficient to provide from about 2-500 mg perdose.

In one embodiment, compounds of the invention are administered incombination with a chymase inhibitor, such as TPC-806 and2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[{3,4-dioxo-1-phenyl-7-(2-pyridyloxy)}-2-heptyl]acetamide(NK3201).

In one embodiment, compounds of the invention are administered incombination with a diuretic. Representative diuretics include, but arenot limited to: carbonic anhydrase inhibitors such as acetazolamide anddichlorphenamide; loop diuretics, which include sulfonamide derivativessuch as acetazolamide, ambuside, azosemide, bumetanide, butazolamide,chloraminophenamide, clofenamide, clopamide, clorexolone, disulfamide,ethoxzolamide, furosemide, mefruside, methazolamide, piretanide,torsemide, tripamide, and xipamide, as well as non-sulfonamide diureticssuch as ethacrynic acid and other phenoxyacetic acid compounds such astienilic acid, indacrinone and quincarbate; osmotic diuretics such asmannitol; potassium-sparing diuretics, which include aldosteroneantagonists such as spironolactone, and Na⁺ channel inhibitors such asamiloride and triamterene; thiazide and thiazide-like diuretics such asalthiazide, bendroflumethiazide, benzylhydrochlorothiazide,benzthiazide, buthiazide, chlorthalidone, chlorothiazide,cyclopenthiazide, cyclothiazide, epithiazide, ethiazide, fenquizone,flumethiazide, hydrochlorothiazide, hydroflumethiazide, indapamide,methylclothiazide, meticrane, metolazone, paraflutizide, polythiazide,quinethazone, teclothiazide, and trichloromethiazide; and combinationsthereof. In a particular embodiment, the diuretic is selected fromamiloride, bumetanide, chlorothiazide, chlorthalidone, dichlorphenamide,ethacrynic acid, furosemide, hydrochlorothiazide, hydroflumethiazide,indapamide, methylclothiazide, metolazone, torsemide, triamterene, andcombinations thereof. The diuretic will be administered in an amountsufficient to provide from about 5-50 mg per day, more typically 6-25 mgper day, with common dosages being 6.25 mg, 12.5 mg or 25 mg per day.

Compounds of the invention may also be administered in combination withan endothelin converting enzyme (ECE) inhibitor, examples of whichinclude, but are not limited to, phosphoramidon, CGS 26303, andcombinations thereof.

In a particular embodiment, compounds of the invention are administeredin combination with an endothelin receptor antagonist. Representativeendothelin receptor antagonists include, but are not limited to:selective endothelin receptor antagonists that affect endothelin Areceptors, such as avosentan, ambrisentan, atrasentan, BQ-123,clazosentan, darusentan, sitaxentan, and zibotentan; and dual endothelinreceptor antagonists that affect both endothelin A and B receptors, suchas bosentan, macitentan, tezosentan).

In yet another embodiment, a compound of the invention is administeredin combination with one or more HMG-CoA reductase inhibitors, which arealso known as statins. Representative statins include, but are notlimited to, atorvastatin, fluvastatin, lovastatin, pitavastatin,pravastatin, rosuvastatin and simvastatin.

In one embodiment, compounds of the invention are administered incombination with a monoamine reuptake inhibitor, examples of whichinclude, by way of illustration and not limitation, norepinephrinereuptake inhibitors such as atomoxetine, buproprion and the buproprionmetabolite hydroxybuproprion, maprotiline, reboxetine, and viloxazine;selective serotonin reuptake inhibitors (SSRIs) such as citalopram andthe citalopram metabolite desmethylcitalopram, dapoxetine, escitalopram(e.g., escitalopram oxalate), fluoxetine and the fluoxetine desmethylmetabolite norfluoxetine, fluvoxamine (e.g., fluvoxamine maleate),paroxetine, sertraline and the sertraline metabolite demethylsertraline;dual serotonin-norepinephrine reuptake inhibitors (SNRIs) such asbicifadine, duloxetine, milnacipran, nefazodone, and venlafaxine; andcombinations thereof.

In another embodiment, compounds of the invention are administered incombination with a muscle relaxant, examples of which include, but arenot limited to: carisoprodol, chlorzoxazone, cyclobenzaprine,diflunisal, metaxalone, methocarbamol, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a natriuretic peptide or analog, examples of whichinclude but are not limited to: carperitide, CD-NP (Nile Therapeutics),CU-NP, nesiritide, PL-3994 (Palatin Technologies, Inc.), ularitide,cenderitide, and compounds described in Ogawa et al (2004) J. Biol.Chem. 279:28625-31. These compounds are also referred to as natriureticpeptide receptor-A (NPR-A) agonists. In another embodiment, compounds ofthe invention are administered in combination with a natriuretic peptideclearance receptor (NPR-C) antagonist such as SC-46542, cANF (4-23), andAP-811 (Veale (2000) Bioorg Med Chem Lett 10:1949-52). For example,AP-811 has shown synergy when combined with the NEP inhibitor, thiorphan(Wegner (1995) Clin. Exper. Hypert. 17:861-876).

In another embodiment, compounds of the invention are administered incombination with a neprilysin (NEP) inhibitor. Representative NEPinhibitors include, but are not limited to: AHU-377; candoxatril;candoxatrilat; dexecadotril((+)-N-[2(R)-(acetylthiomethyl)-3-phenylpropionyl]glycine benzyl ester);CGS-24128(3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionicacid); CGS-24592((S)-3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionicacid); CGS-25155(N-[9(R)-(acetylthiomethyl)-10-oxo-1-azacyclodecan-2(S)-ylcarbonyl]-4(R)-hydroxy-L-prolinebenzyl ester); 3-(1-carbamoylcyclohexyl)propionic acid derivativesdescribed in WO 2006/027680 to Hepworth et al. (Pfizer Inc.); JMV-390-1(2(R)-benzyl-3-(N-hydroxycarbamoyl)propionyl-L-isoleucyl-L-leucine);ecadotril; phosphoramidon; retrothiorphan; RU-42827(2-(mercaptomethyl)-N-(4-pyridinyl)benzenepropionamide); RU-44004(N-(4-morpholinyl)-3-phenyl-2-(sulfanylmethyl)propionamide); SCH-32615((S)—N—[N-(1-carboxy-2-phenylethyl)-L-phenylalanyl]-β-alanine) and itsprodrug SCH-34826((S)—N—[N-[1-[[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-β-alanine);sialorphin; SCH-42495(N-[2(S)-(acetylsulfanylmethyl)-3-(2-methylphenyl)propionyl]-L-methionineethyl ester); spinorphin; SQ-28132(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]leucine); SQ-28603(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-β-alanine); SQ-29072(7-[[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]amino]heptanoic acid);thiorphan and its prodrug racecadotril; UK-69578(cis-4-[[[1-[2-carboxy-3-(2-methoxyethoxy)propyl]cyclopentyl]carbonyl]amino]cyclohexanecarboxylicacid); UK-447,841(2-{1-[3-(4-chlorophenyl)propylcarbamoyl]-cyclopentylmethyl}-4-methoxybutyricacid); UK-505,749((R)-2-methyl-3-{1-[3-(2-methylbenzothiazol-6-yl)propylcarbamoyl]cyclopentyl}propionicacid); 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoicacid and 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoicacid ethyl ester (WO 2007/056546); daglutril[(3S,2′R)-3-{1-[2′-(ethoxycarbonyl)-4′-phenylbutyl]-cyclopentan-1-carbonylamino}-2,3,4,5-tetrahydro-2-oxo-1H-1-benzazepine-1-aceticacid] described in WO 2007/106708 to Khder et al. (Novartis AG); andcombinations thereof. In a particular embodiment, the NEP inhibitor isselected from AHU-377, candoxatril, candoxatrilat, CGS-24128,phosphoramidon, SCH-32615, SCH-34826, SQ-28603, thiorphan, andcombinations thereof. In a particular embodiment, the NEP inhibitor is acompound such as daglutril or CGS-26303 ([N-[2-(biphenyl-4-yl)-1(S)-(1H-tetrazol-5-yl)ethyl]amino]methylphosphonic acid), which haveactivity both as inhibitors of the endothelin converting enzyme (ECE)and of NEP. Other dual acting ECE/NEP compounds can also be used. TheNEP inhibitor will be administered in an amount sufficient to providefrom about 20-800 mg per day, with typical daily dosages ranging from50-700 mg per day, more commonly 100-600 or 100-300 mg per day.

In one embodiment, compounds of the invention are administered incombination with a nitric oxide donor, examples of which include, butare not limited to nicorandil; organic nitrates such as pentaerythritoltetranitrate; and sydnonimines such as linsidomine and molsidomine.

In yet another embodiment, compounds of the invention are administeredin combination with a non-steroidal anti-inflammatory agent (NSAID).Representative NSAIDs include, but are not limited to: acemetacin,acetyl salicylic acid, alclofenac, alminoprofen, amfenac, amiprilose,aloxiprin, anirolac, apazone, azapropazone, benorilate, benoxaprofen,bezpiperylon, broperamole, bucloxic acid, carprofen, clidanac,diclofenac, diflunisal, diftalone, enolicam, etodolac, etoricoxib,fenbufen, fenclofenac, fenclozic acid, fenoprofen, fentiazac, feprazone,flufenamic acid, flufenisal, fluprofen, flurbiprofen, furofenac,ibufenac, ibuprofen, indomethacin, indoprofen, isoxepac, isoxicam,ketoprofen, ketorolac, lofemizole, lornoxicam, meclofenamate,meclofenamic acid, mefenamic acid, meloxicam, mesalamine, miroprofen,mofebutazone, nabumetone, naproxen, niflumic acid, oxaprozin, oxpinac,oxyphenbutazone, phenylbutazone, piroxicam, pirprofen, pranoprofen,salsalate, sudoxicam, sulfasalazine, sulindac, suprofen, tenoxicam,tiopinac, tiaprofenic acid, tioxaprofen, tolfenamic acid, tolmetin,triflumidate, zidometacin, zomepirac, and combinations thereof. In aparticular embodiment, the NSAID is selected from etodolac,flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meloxicam,naproxen, oxaprozin, piroxicam, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with an N-methyl d-aspartate (NMDA) receptor antagonist,examples of which include, by way of illustration and not limitation,including amantadine, dextromethorphan, dextropropoxyphene, ketamine,ketobemidone, memantine, methadone, and so forth.

In still another embodiment, compounds of the invention are administeredin combination with an opioid receptor agonist (also referred to asopioid analgesics). Representative opioid receptor agonists include, butare not limited to: buprenorphine, butorphanol, codeine, dihydrocodeine,fentanyl, hydrocodone, hydromorphone, levallorphan, levorphanol,meperidine, methadone, morphine, nalbuphine, nalmefene, nalorphine,naloxone, naltrexone, nalorphine, oxycodone, oxymorphone, pentazocine,propoxyphene, tramadol, and combinations thereof. In certainembodiments, the opioid receptor agonist is selected from codeine,dihydrocodeine, hydrocodone, hydromorphone, morphine, oxycodone,oxymorphone, tramadol, and combinations thereof.

In a particular embodiment, compounds of the invention are administeredin combination with a phosphodiesterase (PDE) inhibitor, particularly aPDE-V inhibitor. Representative PDE-V inhibitors include, but are notlimited to, avanafil, lodenafil, mirodenafil, sildenafil (Revatio®),tadalafil (Adcirca®), vardenafil (Levitra®), and udenafil.

In another embodiment, compounds of the invention are administered incombination with a prostaglandin analog (also referred to as prostanoidsor prostacyclin analogs). Representative prostaglandin analogs include,but are not limited to, beraprost sodium, bimatoprost, epoprostenol,iloprost, latanoprost, tafluprost, travoprost, and treprostinil, withbimatoprost, latanoprost, and tafluprost being of particular interest.

In yet another embodiment, compounds of the invention are administeredin combination with a prostaglandin receptor agonist, examples of whichinclude, but are not limited to, bimatoprost, latanoprost, travoprost,and so forth.

Compounds of the invention may also be administered in combination witha renin inhibitor, examples of which include, but are not limited to,aliskiren, enalkiren, remikiren, and combinations thereof.

In another embodiment, compounds of the invention are administered incombination with a selective serotonin reuptake inhibitor (SSRI).Representative SSRIs include, but are not limited to: citalopram and thecitalopram metabolite desmethylcitalopram, dapoxetine, escitalopram(e.g., escitalopram oxalate), fluoxetine and the fluoxetine desmethylmetabolite norfluoxetine, fluvoxamine (e.g., fluvoxamine maleate),paroxetine, sertraline and the sertraline metabolite demethylsertraline,and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a 5-HT_(1D) serotonin receptor agonist, examples ofwhich include, by way of illustration and not limitation, triptans suchas almotriptan, avitriptan, eletriptan, frovatriptan, naratriptan,rizatriptan, sumatriptan, and zolmitriptan.

In one embodiment, compounds of the invention are administered incombination with a sodium channel blocker, examples of which include, byway of illustration and not limitation, carbamazepine, fosphenytoin,lamotrigine, lidocaine, mexiletine, oxcarbazepine, phenytoin, andcombinations thereof.

In one embodiment, compounds of the invention are administered incombination with a soluble guanylate cyclase stimulator or activator,examples of which include, but are not limited to ataciguat, riociguat,and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a tricyclic antidepressant (TCA), examples of whichinclude, by way of illustration and not limitation, amitriptyline,amitriptylinoxide, butriptyline, clomipramine, demexiptiline,desipramine, dibenzepin, dimetacrine, dosulepin, doxepin, imipramine,imipraminoxide, lofepramine, melitracen, metapramine, nitroxazepine,nortriptyline, noxiptiline, pipofezine, propizepine, protriptyline,quinupramine, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a vasopressin receptor antagonist, examples of whichinclude, by way of illustration and not limitation, conivaptan andtolvaptan.

Combined secondary therapeutic agents may also be helpful in furthercombination therapy with compounds of the invention. For example,compounds of the invention can be combined with a diuretic and an ARB,or a calcium channel blocker and an ARB, or a diuretic and an ACEinhibitor, or a calcium channel blocker and a statin. Specific examplesinclude, a combination of the ACE inhibitor enalapril (in the maleatesalt form) and the diuretic hydrochlorothiazide, which is sold under themark Vaseretic®, or a combination of the calcium channel blockeramlodipine (in the besylate salt form) and the ARB olmesartan (in themedoxomil prodrug form), or a combination of a calcium channel blockerand a statin, all may also be used with the compounds of the invention.Other therapeutic agents such as α₂-adrenergic receptor agonists andvasopressin receptor antagonists may also be helpful in combinationtherapy. Exemplary a₂-adrenergic receptor agonists include clonidine,dexmedetomidine, and guanfacine.

The following formulations illustrate representative pharmaceuticalcompositions of the invention.

Exemplary Hard Gelatin Capsules for Oral Administration

A compound of the invention (50 g), 440 g spray-dried lactose and 10 gmagnesium stearate are thoroughly blended. The resulting composition isthen loaded into hard gelatin capsules (500 mg of composition percapsule). Alternately, a compound of the invention (20 mg) is thoroughlyblended with starch (89 mg), microcrystalline cellulose (89 mg) andmagnesium stearate (2 mg). The mixture is then passed through a No. 45mesh U.S. sieve and loaded into a hard gelatin capsule (200 mg ofcomposition per capsule).

Alternately, a compound of the invention (30 g), a secondary agent (20g), 440 g spray-dried lactose and 10 g magnesium stearate are thoroughlyblended, and processed as described above.

Exemplary Gelatin Capsule Formulation for Oral Administration

A compound of the invention (100 mg) is thoroughly blended withpolyoxyethylene sorbitan monooleate (50 mg) and starch powder (250 mg).The mixture is then loaded into a gelatin capsule (400 mg of compositionper capsule). Alternately, a compound of the invention (70 mg) and asecondary agent (30 mg) are thoroughly blended with polyoxyethylenesorbitan monooleate (50 mg) and starch powder (250 mg), and theresulting mixture loaded into a gelatin capsule (400 mg of compositionper capsule).

Alternately, a compound of the invention (40 mg) is thoroughly blendedwith microcrystalline cellulose (Avicel PH 103; 259.2 mg) and magnesiumstearate (0.8 mg). The mixture is then loaded into a gelatin capsule(Size #1, White, Opaque) (300 mg of composition per capsule).

Exemplary Tablet Formulation for Oral Administration

A compound of the invention (10 mg), starch (45 mg) and microcrystallinecellulose (35 mg) are passed through a No. 20 mesh U.S. sieve and mixedthoroughly. The granules so produced are dried at 50-60° C. and passedthrough a No. 16 mesh U.S. sieve. A solution of polyvinylpyrrolidone (4mg as a 10% solution in sterile water) is mixed with sodiumcarboxymethyl starch (4.5 mg), magnesium stearate (0.5 mg), and talc (1mg), and this mixture is then passed through a No. 16 mesh U.S. sieve.The sodium carboxymethyl starch, magnesium stearate and talc are thenadded to the granules. After mixing, the mixture is compressed on atablet machine to afford a tablet weighing 100 mg.

Alternately, a compound of the invention (250 mg) is thoroughly blendedwith microcrystalline cellulose (400 mg), silicon dioxide fumed (10 mg),and stearic acid (5 mg). The mixture is then compressed to form tablets(665 mg of composition per tablet).

Alternately, a compound of the invention (400 mg) is thoroughly blendedwith cornstarch (50 mg), croscarmellose sodium (25 mg), lactose (120mg), and magnesium stearate (5 mg). The mixture is then compressed toform a single-scored tablet (600 mg of composition per tablet).

Alternately, a compound of the invention (100 mg) is thoroughly blendedwith cornstarch (100 mg) with an aqueous solution of gelatin (20 mg).The mixture is dried and ground to a fine powder. Microcrystallinecellulose (50 mg) and magnesium stearate (5 mg) are then admixed withthe gelatin formulation, granulated and the resulting mixture compressedto form tablets (100 mg of the compound of the invention per tablet).

Exemplary Suspension Formulation for Oral Administration

The following ingredients are mixed to form a suspension containing 100mg of the compound of the invention per 10 mL of suspension:

Ingredients Amount Compound of the invention 1.0 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum ® K(magnesium aluminum silicate) 1.0 g Flavoring 0.035 mL Colorings 0.5 mgDistilled water q.s. to 100 mL

Exemplary Liquid Formulation for Oral Administration

A suitable liquid formulation is one with a carboxylic acid-based buffersuch as citrate, lactate and maleate buffer solutions. For example, acompound of the invention (which may be pre-mixed with DMSO) is blendedwith a 100 mM ammonium citrate buffer and the pH adjusted to pH 5, or isblended with a 100 mM citric acid solution and the pH adjusted to pH 2.Such solutions may also include a solubilizing excipient such as acyclodextrin, for example the solution may include 10 wt %hydroxypropyl-β-cyclodextrin.

Other suitable formulations include a 5% NaHCO₃ solution, with orwithout cyclodextrin.

Exemplary Injectable Formulation for Administration by Injection

A compound of the invention (0.2 g) is blended with 0.4 M sodium acetatebuffer solution (2.0 mL). The pH of the resulting solution is adjustedto pH 4 using 0.5 N aqueous hydrochloric acid or 0.5 N aqueous sodiumhydroxide, as necessary, and then sufficient water for injection isadded to provide a total volume of 20 mL. The mixture is then filteredthrough a sterile filter (0.22 micron) to provide a sterile solutionsuitable for administration by injection.

Exemplary Compositions for Administration by Inhalation

A compound of the invention (0.2 mg) is micronized and then blended withlactose (25 mg). This blended mixture is then loaded into a gelatininhalation cartridge. The contents of the cartridge are administeredusing a dry powder inhaler, for example.

Alternately, a micronized compound of the invention (10 g) is dispersedin a solution prepared by dissolving lecithin (0.2 g) in demineralizedwater (200 mL). The resulting suspension is spray dried and thenmicronized to form a micronized composition comprising particles havinga mean diameter less than about 1.5 μm. The micronized composition isthen loaded into metered-dose inhaler cartridges containing pressurized1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 10 μgto about 500 μg of the compound of the invention per dose whenadministered by the inhaler.

Alternately, a compound of the invention (25 mg) is dissolved in citratebuffered (pH 5) isotonic saline (125 mL). The mixture is stirred andsonicated until the compound is dissolved. The pH of the solution ischecked and adjusted, if necessary, to pH 5 by slowly adding aqueous 1 NNaOH. The solution is administered using a nebulizer device thatprovides about 10 μg to about 500 μg of the compound of the inventionper dose.

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments of the invention. These specific embodiments,however, are not intended to limit the scope of the invention in any wayunless specifically indicated.

The following abbreviations have the following meanings unless otherwiseindicated and any other abbreviations used herein and not defined havetheir standard, generally accepted meaning:

-   AcOH acetic acid-   BOC t-butoxycarbonyl (—C(O)OC(CH₃)₃)-   (BOC)₂O di-t-butyl dicarbonate-   DCC dicyclohexylcarbodiimide-   DCM dichloromethane or methylene chloride-   DIPEA N,N-diisopropylethylamine-   DMA N,N-dimethylacetamide-   DMAP 4-dimethylaminopyridine-   DMF N,N-dimethylformamide-   EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-   Et₃N triethylamine-   Et₂O diethyl ether-   Et₃SiH triethylsilane-   EtOAc ethyl acetate-   EtOH ethanol-   HATU N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium    hexafluorophosphate-   HCTU 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium    hexafluorophosphate-   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-   HOBt 1-hydroxybenzotriazole-   MeCN acetonitrile-   MeOH methanol-   NaHMDS sodium hexamethyldisilazide-   Pd(dppf)₂Cl₂ 1,1-bis(diphenylphosphino) ferrocene palladium chloride-   Pd(PPh₃)₄ tetrakis(triphenylphosphine)palladium(0)-   PE petroleum ether-   PMB p-methoxybenzyl-   PyBOP benzotriazol-1-yloxytris(pyrrolidino)phosphonium    hexafluorophosphate-   SilicaCat®DPP-Pd silica based diphenylphosphine palladium (II)    catalyst-   TFA trifluoroacetic acid-   THF tetrahydrofuran-   TMSCl Trimethylsilyl chloride-   Tr trityl

Unless noted otherwise, all materials, such as reagents, startingmaterials and solvents, were purchased from commercial suppliers (suchas Sigma-Aldrich, Fluka Riedel-de Haën, and the like) and were usedwithout further purification.

Reactions were run under nitrogen atmosphere, unless noted otherwise.The progress of reactions were monitored by thin layer chromatography(TLC), analytical high performance liquid chromatography (anal. HPLC),and mass spectrometry, the details of which are given in specificexamples. Solvents used in analytical HPLC were as follows: solvent Awas 98% H₂O/2% MeCN/1.0 mL/L TFA; solvent B was 90% MeCN/10% H₂O/1.0mL/L TFA.

Reactions were worked up as described specifically in each preparationfor example; commonly reaction mixtures were purified by extraction andother purification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by preparative HPLC, typically using Microsorb C18and Microsorb BDS column packings and conventional eluents. Progress ofreactions was typically measured by liquid chromatography massspectrometry (LCMS). Characterization of isomers were done by NuclearOverhauser effect spectroscopy (NOE). Characterization of reactionproducts was routinely carried out by mass and ¹H-NMR spectrometry. ForNMR measurement, samples were dissolved in deuterated solvent (CD₃OD,CDCl₃, or DMSO-d₆), and ¹H-NMR spectra were acquired with a VarianGemini 2000 instrument (400 MHz) under standard observation conditions.Mass spectrometric identification of compounds was typically conductedusing an electrospray ionization method (ESMS) with an AppliedBiosystems (Foster City, Calif.) model API 150 EX instrument or anAgilent (Palo Alto, Calif.) model 1200 LC/MSD instrument.

Preparation 1: (S)-2-(4-Bromobenzyl)-5-oxopyrrolidine-1-carboxylic Acidt-Butyl Ester

To a solution of (R)-2-amino-3-(4-bromophenyl)propionic acid (50 g, 0.2mol) in MeCN (700 mL) was added a solution of NaOH (16.4 g, 0.4 mol) inwater (700 mL) at −5° C. After stirring for 10 minutes, a solution of(BOC)₂O (44.7 g, 0.2 mol) in MeCN (100 mL) was added. The mixture waswarmed to room temperature and stirred overnight. After the evaporationof the MeCN, the residue was diluted with DCM (800 mL) and acidifiedwith 1 M HCl to pH 2 at −5° C. The aqueous was extracted with DCM (3×200mL). The combined organic layers were washed with saturated aqueous NaCl(500 mL), dried over Na₂SO₄ and concentrated to yield Compound 1 (66.5g) as a white solid. LC-MS: 366 [M+Na], 709 [2M+Na].

To a solution of Compound 1 (66.5 g, 193 μmol), Meldrum's acid (33.4 g,232 mmol) and DMAP (37.7 g, 309 mmol) in anhydrous DCM (600 mL), wasadded dropwise a solution of DCC (47.9 g, 232 mmol) in anhydrous DCM(200 mL) over 1 hour at −5° C. under nitrogen. The mixture was stirredat −5° C. for 8 hours, then refrigerated overnight. Crystals ofdicyclohexylurea were observed. The mixture was filtered, washed with 5%KHSO₄ (5×200 mL) and saturated aqueous NaCl (200 mL), then dried overanhydrous MgSO₄ under refrigeration overnight. The solution was thenevaporated to yield crude Compound 2 (91 g) as a light yellow solid.LC-MS: 492 [M+Na], 961 [2M+Na].

To a solution of crude Compound 2 (91 g, 193 mmol) in anhydrous DCM (1L) was added AcOH (127.5 g, 2.1 mol) at −5° C. under nitrogen. Themixture was stirred at −5° C. for 30 minutes, then NaBH₄ (18.3 g, 483mmol) was added in small portions over 1 hour. After stirring foranother 1 hour at −5° C., saturated aqueous NaCl (500 mL) was added. Theorganic layer was washed with saturated aqueous NaCl (2×300 mL) andwater (2×300 mL), dried over MgSO₄, filtered, and concentrated to yieldthe crude product, which was further purified by washing with Et₂O toyield Compound 3 (68 g) as a light yellow solid. LC-MS: 478 [M+Na], 933[2M+Na].

A solution of Compound 3 (68 g, 149 mmol) in anhydrous toluene (500 mL)was refluxed under nitrogen for 3 hours. After evaporation of thesolvent, the residue was purified by chromatography (hexanes:EtOAc=10:1)to yield the title compound (38 g) as a light yellow oil. LC-MS: 376[M+Na], 729 [2M+Na].

Preparation 2:(3R,5R)-5-(3′-Chlorobiphenyl-4-ylmethyl)-1-(2,2-dimethylpropionyl)-3-hydroxypyrrolidin-2-one

To a solution of (S)-2-(4-bromobenzyl)-5-oxopyrrolidine-1-carboxylicacid t-butyl ester (15 g, 43 mmol) in 1,4-dioxane (600 mL) was added3-chlorophenylboronic acid (8 g, 51 mmol) and Pd(dppf)₂Cl₂ (3.1 g, 4.2mmol) at room temperature under nitrogen. After stirring for 10 minutes,a solution of K₂CO₃ (11.7 g, 85 mmol) in water (60 mL) was added. Themixture was heated to 60° C. and stirred overnight. After evaporation ofthe solvent, water (200 mL) was added and extracted with EtOAc (3×200mL). The combined organic layers were washed with saturated aqueous NaCl(400 mL), dried over Na₂SO₄, and concentrated to yield the crude productwhich was further purified by column chromatography (hexanes:EtOAc=6:1)to yield Compound 1 (15 g) as a light yellow solid. LC-MS: 408 [M+Na].

To a solution of Compound 1 (15 g, 0.039 mol) in anhydrous DCM (250 mL)was added TFA (20 mL, 270 mmol) at −5° C. under nitrogen. The mixturewas warmed to room temperature and stirred overnight. After evaporationof the solvent, the residue was diluted with EtOAc (300 mL), then washedwith saturated aqueous NaHCO₃ (3×200 mL), water (200 mL), saturatedaqueous NaCl (250 mL), then dried over Na₂SO₄ and concentrated to yieldcrude Compound 2 (11 g) as a light yellow solid. LC-MS: 286 [M+H].

To a solution of NaH (2.3 g, 98 mmol) in anhydrous THF (200 mL) wasadded dropwise a solution of Compound 2 (11 g, 39 mmol) in anhydrous THF(100 mL) over 30 minutes at 0° C. under nitrogen. The mixture was warmedto room temperature and stirred for 2 hours. After cooling to 0° C.,pivaloyl chloride (6 g, 51 mmol) was added dropwise over 30 minutes. Themixture was warmed to room temperature and stirred overnight. Thereaction was quenched with saturated aqueous NH₄Cl (200 mL) andextracted with EtOAc (3×200 mL). The combined organic layers were washedwith saturated aqueous NaCl (300 mL), dried over MgSO₄, filtered, andconcentrated to yield the crude product which was further purified bychromatography (hexanes:EtOAc=25:1) to yield Compound 3 (10.5 g) as alight yellow solid. LC-MS: 391 [M+Na].

To a solution of Compound 3 (10.5 g, 29 mmol) in anhydrous THF (120 mL)was added dropwise NaHMDS (29 mL, 58 mmol) over 30 minutes at −78° C.under nitrogen. After stirring at −78° C. for 90 minutes, a solution of(+)-(8,8-dichlorocamphorylsulfonyl)oxaziridine (15.6 g, 52 mmol) wasadded dropwise over 30 minutes. After stirring at −78° C. for 2 hours,the reaction was quenched with saturated NH₄Cl (400 mL) and extractedwith EtOAc (3×300 mL). The combined organic layers were washed withsaturated aqueous NaCl (300 mL), dried over MgSO₄, filtered, andconcentrated to give the crude product which was further purified bychromatography (hexanes:EtOAc=15:1) to yield the title compound (9.6 g)as a light yellow solid. LC-MS: 408 [M+Na].

Preparation 3:(2R,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic Acid EthylEster

A solution of(3R,5R)-5-(3′-chlorobiphenyl-4-ylmethyl)-1-(2,2-dimethylpropionyl)-3-hydroxypyrrolidin-2-one(9.6 g, 25 mmol) in concentrated HCl (81 mL, 81 mmol) was heated at 100°C. for 16 hours. The mixture was then concentrated to give the crudeproduct which was further purified by washing with Et₂O to yieldCompound 1 (5.7 g) as a light yellow solid HCl salt. LC-MS: 320 [M+H].

To a solution of Compound 1 (5.7 g, 18 mmol) in EtOH (10 mL) was added8M HCl in EtOH (120 mL, 960 mmol) at room temperature. The mixture washeated at 50° C. for 16 hours. After concentration, the crude productwas further purified by washing with Et₂O to yield the title compound(2.1 g) as a light yellow solid HCl salt. LC-MS: 348 [M+H].

Preparation 4:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicAcid Ethyl Ester

HATU (328 mg, 862 μmol) was added to a solution of1-hydroxy-1H-1,2,3-benzotriazole-6-carboxylic acid (154 mg, 862 μmol) inDMF (1.5 mL, 19.5 mmol) and the resulting mixture was stirred at roomtemperature for 10 minutes. DIPEA (0.3 mL, 1.7 mmol) was added to themixture, followed by(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (200 mg, 575 μmol) and the mixture was stirred at room temperaturefor 1 hour. The mixture was concentrated in vacuo and the resultingresidue was purified by reverse phase chromatography (35-80% MeCN inwater with 0.05% TFA) to yield the title compound (197 mg) as a whitesolid.

Preparation 5:(2R,4R)-4-t-Butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicAcid

A solution of(3R,5R)-5-(3′-chlorobiphenyl-4-ylmethyl)-1-(2,2-dimethylpropionyl)-3-hydroxypyrrolidin-2-one(4.5 g, 11.7 mmol) in concentrated HCl (30 mL) was stirred at 100° C.for 16 hours. The mixture was concentrated in vacuo to yield Compound 1(4 g) as a white solid HCl salt. LC-MS: 321 [M+H]⁺.

To a solution of NaOH (1.8 g, 45.2 mmol) in water (100 mL), was addedCompound 1 (4 g, 11.3 mmol) in MeCN (100 mL) dropwise. The mixture wasstirred for 10 minutes at 0° C. Di-t-butyldicarbonate (7.17 g, 33.8mmol) was added and the mixture was stirred for 15 hours at roomtemperature. The resulting mixture was concentrated in vacuo to removeMeCN, then diluted with DCM (300 mL), and the pH adjusted to pH=5-6 with1N aqueous HCl. Then the organic layer was collected and the residue wasextracted with DCM (3×300 mL). The combined organic layers wereconcentrated and washed with hexanes (150 mL) to yield the titlecompound (4 g) as a white solid. LC-MS: 442 [M+Na]⁺.

Preparation 6:(2R,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic Acid2,2,3,3,3-pentafluoropropyl Ester

To a solution of(2R,4R)-4-t-butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicacid (0.9 g, 6 mmol) and 2,2,3,3,3-pentafluoropropan-1-ol (450 mg, 3mmol) in DCM (30 mL) was added DCC (880 mg, 4.3 mmol) and DMAP (260 mg,2.1 mmol). The resulting mixture was stirred for 15 hours at roomtemperature, then concentrated in vacuo. The residue was dissolved inEtOAc (100 mL) and washed with water (30 mL) and saturated aqueous NaCl(30 mL). The organic layer was collected and concentrated and purifiedby column chromatography (hexanes/EtOAc=5:1) to yield Compound 1 (0.4 g)as a white solid. LC-MS: 574 [M+Na]⁺.

A solution of Compound 1 (0.4 g, 690 μmol) in 1.4 M HCl in a 1,4-dioxanesolution (15 mL) was stirred overnight, and then concentrated in vacuo.The residue was dispersed in EtOAc (10 mL), and the precipitate wascollected by filtration to yield the title compound as an off-whitesolid HCl salt (165 mg). LC-MS: 452 [M+H]⁺. ¹H NMR: (DMSO-d₆) 1.95-1.82(m, 2H), 2.99-2.98 (m, 2H), 3.56 (br, 1H), 4.41-4.38 (m, 1H), 4.92-4.82(m, 2H), 6.35 (s, 1H), 7.71-7.38 (m, 8H), 8.09 (s, 3H).

Preparation 7:(2R,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic Acid5-Methyl-2-oxo[1,3]dioxol-4-ylmethyl Ester

A suspension of(2R,4R)-4-t-butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicacid (740 mg, 1.8 mmol), 4-(bromomethyl)-5-methyl-1,3-dioxol-2-one (340mg, 1.8 mmol), potassium iodide (58 mg, 350 μmol), and K₂CO₃ (486 mg,3.5 mmol) in DMF (20 mL) was stirred for 4 hours at room temperature.The mixture was diluted with EtOAc (150 mL) and washed with water (30mL). The organic layer was collected and concentrated and purified bycolumn chromatography (hexanes/EtOAc=1:1) to yield a white solid (490mg). LC-MS: 554 [M+23]⁺. A solution of this solid (476 mg, 890 μmol) in3 N HCl in 1,4-dioxane (20 mL) was stirred overnight, and thenconcentrated in vacuo. The residue was dispersed in EtOAc (10 mL), andthe precipitate was collected by filtration to yield the title compoundas an off-white solid (290 mg). LC-MS: 432 [M+H]⁺. ¹H NMR: (DMSO-d₆)1.92-1.82 (m, 2H), 2.16 (s, 3H), 2.99 (br, 2H), 3.56 (br, 1H), 4.35-4.32(m, 1H), 5.017 (s, 2H), 6.17 (s, 1H), 7.39-7.36 (m, 4H), 7.71-7.68 (m,4H), 8.05 (s, 3H).

Preparation 8:(2R,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic AcidButyryloxymethyl Ester

A solution of(2R,4R)-4-t-butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicacid (900 mg, 2.1 mmol), chloromethyl butyrate (350 mg, 2.6 mmol),sodium iodide (481 mg, 3.21 mmol) and DIPEA (828 mg, 6.42 mmol) in DMF(20 mL) was stirred for 16 hours at 30° C. The mixture was diluted withEtOAc (150 mL) and washed with water (50 mL) and saturated aqueous NaCl(50 mL). The organic layer was collected and concentrated and purifiedby column chromatography (hexanes/EtOAc=5:1) to yield a white solid (240mg). LC-MS: 542 [M+Na]⁺. A solution of this solid (240 mg, 460 μmol) in1.4 M HCl in 1,4-dioxane (15 mL) was stirred overnight, and thenconcentrated in vacuo. The residue was dispersed in EtOAc (10 mL), andthe precipitated was collected by filtration to yield the title compoundas an off-white solid HCl salt (140 mg). LC-MS: 420 [M+H]⁺. ¹H NMR:(DMSO) 0.85 (t, J=7.5 Hz, 3H), 1.61-1.52 (m, 2H), 1.89-1.86 (m, 2H),2.30 (t, J=7.5 Hz, 2H), 2.98 (br, 2H), 3.56 (br, 1H), 4.33-4.30 (m, 1H),5.74-5.68 (m, 2H), 6.21 (s, 1H), 7.37-7.35 (m, 4H), 7.70-7.767 (m, 4H),8.01 (brs, 3H).

Preparation 9: (2R,4R)-4-Amino-5-(4-bromophenyl)-2-hydroxypentanoic AcidEthyl Ester

To a solution of (S)-2-(4-bromobenzyl)-5-oxopyrrolidine-1-carboxylicacid t-butyl ester (38 g, 107 mmol) in anhydrous DCM (250 mL) was addedTFA (20 mL, 0.27 mol) at −5° C. under nitrogen. The mixture was warmedto room temperature and stirred overnight. After evaporation of thesolvent, the residue was diluted with EtOAc (300 mL) and washed withsaturated aqueous NaHCO₃ (3×200 mL), water (200 mL), saturated aqueousNaCl (250 mL), dried over Na₂SO₄ and concentrated to yield crudeCompound 1 (24 g) as a light yellow solid. LC-MS: 254 [M+H].

To a solution of NaH (8.6 g, 250 mmol) in anhydrous THF (200 mL) wasadded dropwise a solution of Compound 1 (24 g, 94 mmol) in anhydrous THF(200 mL) over 30 minutes at 0° C. under nitrogen. The mixture was warmedto room temperature and stirred for 2 hours. After cooling to 0° C.,pivaloyl chloride (18 g, 150 mmol) was added dropwise over 30 minutes.The mixture was warmed to room temperature and stirred overnight. Thereaction was quenched with saturated aqueous NH₄Cl (300 mL) andextracted with EtOAc (3×200 mL). The combined organic layers were washedwith saturated aqueous NaCl (300 mL), dried over MgSO₄, filtered andconcentrated to yield the crude product, which was further purified bychromatography (hexanes:EtOAc=25:1) to yield Compound 2 (18 g) as alight yellow solid. LC-MS: 360 [M+Na].

To a solution of Compound 2 (18 g, 53 mmol) in anhydrous THF (250 mL)was added dropwise NaHMDS (47.7 mL, 96 mmol) over 30 minutes at −78° C.under nitrogen. After stirring at −78° C. for 90 minutes, a solution of(+)-(8,8-dichlorocamphorylsulfonyl)-oxaziridine (31.6 g, 106 mmol) wasadded dropwise over 30 minutes. After stirring at −78° C. for 2 hours,the reaction was quenched with saturated aqueous NH₄Cl (400 mL) andextracted with EtOAc (3×300 mL). The combined organic layers were washedwith saturated aqueous NaCl (300 mL), dried over MgSO₄, filtered, andconcentrated to give the crude product which was further purified bychromatography (hexanes:EtOAc=15:1) to yield Compound 3 (8.9 g) as alight yellow solid. LC-MS: 376 [M+Na].

A solution of Compound 3 (8.9 g, 25 mmol) in concentrated HCl (81 mL, 81mmol) was heated at 100° C. for 16 hours. The mixture was thenconcentrated to yield the crude product which was further purified bywashing with Et₂O to yield Compound 4 (7 g) as a light yellow solid HClsalt. LC-MS: 323 [M+H].

A solution of Compound 4 (7 g, 22 mmol) in EtOH (10 mL) was combinedwith 8M HCl in EtOH (120 mL, 960 mmol) at room temperature. The mixturewas heated at 50° C. for 16 hours, then concentrated. The crude productwas further purified by washing with Et₂O to yield the title compound (6g) as a light yellow solid HCl salt. LC-MS: 352 [M+H].

Preparation 10: 5-Hydroxy-1-pyridin-2-yl-1H-pyrazole-3-carboxylic Acid

To a suspension of 5-hydroxy-1-pyridin-2-yl-1h-pyrazole-3-carboxylicacid ethyl ester (351.6 mg, 1.5 mmol) in MeOH (5.0 mL, 120 mmol) at roomtemperature was added LiOH monohydrate (126.5 mg, 3.0 mmol), forming aclear solution. The solution was stirred at room temperature overnight.The solution was then concentrated. To the resulting residue was added1N aqueous HCl to achieve pH-2, forming a precipitate. Water (4.0 mL)was added and the resulting mixture was stirred at room temperature for1 hour, then filtered. The solids were rinsed with water and dried undervacuum to yield the title compounds as a white solid (128 mg).

Preparation 11:(2R,4R)-4-Amino-5-(5′-chloro-2′-fluoro-biphenyl-4-yl)-2-hydroxypentanoicAcid

To a solution of (S)-2-(4-bromobenzyl)-5-oxopyrrolidine-1-carboxylicacid t-butyl ester (25 g, 70.6 mmol) in 1,4-dioxane (500 mL) was added5-chloro-2-fluorophenylboronic acid (24.6 g, 141 mmol), Pd(PPh₃)₄ (4.1g, 3.5 mmol) and a solution of K₂CO₃ (17.8 g, 141 mmol) in water (90mL), at room temperature under nitrogen. The mixture was heated to 60°C. and stirred overnight. Water (500 mL) was added and the solventevaporated. The mixture was extracted with EtOAc (3×200 mL). Thecombined organic layers were washed with saturated aqueous NaCl (300 mL)and filtered. The filtrate was concentrated to yield the crude productwhich was purified by chromatography to yield Compound 1 (22.7 g) as alight yellow solid. LC-MS: 829.2 [2M+Na⁺].

To a solution of Compound 1 (4.9 g, 12.1 mol) in DCM (100 mL) was addedTFA (4.5 mL, 60.7 mmol) at 0° C. under nitrogen, and stirred for 1 hour.The mixture was warmed to room temperature for 1.5 hours. Afterevaporation of the solvent, the residue was diluted with EtOAc (100 mL),then washed with saturated aqueous NaHCO₃ (3×100 mL), water (2×100 mL),saturated aqueous NaCl (100 mL), then dried over Na₂SO₄. The mixture wasfiltered and the filtrate was concentrated to yield crude Compound 2(combined with a separate lot for a total of 16.9 g). LC-MS: 304 [M+H].

To a solution of NaH (2.4 g, 695 mmol) in THF (200 mL) was addeddropwise a solution of Compound 2 (8.5 g, 278 mmol) in THF (50 mL) at 0°C. under nitrogen. The mixture was warmed to room temperature andstirred for 2 hours. After cooling to 0° C., pivaloyl chloride (5 g,41.7 mmol) was added dropwise over 30 minutes. The mixture was warmed toroom temperature and stirred for 9.5 hours. The reaction was quenchedwith saturated aqueous NH₄Cl (250 mL) and extracted with EtOAc (3×400mL). The combined organic layers were dried over Na₂SO₄ and concentratedto yield the crude product which was purified by chromatography to yieldCompound 3 (18 g) as a yellow solid. LC-MS: 388 [M+H⁺].

To a solution of Compound 3 (9 g, 23.2 mmol) in THF (200 mL) was addeddropwise NaHMDS (20.9 mL, 41.8 mmol) at −78° C. under nitrogen. Afterstirring for 1 hour at −78° C., a solution of(+)-(8,8-dichlorocamphorylsulfonyl)oxaziridine (10.4 g, 34.8 mmol) inTHF (50 mL) was added dropwise. After stirring at −78° C. for 1 hour,the reaction was quenched with saturated aqueous NH₄Cl (50 mL) andextracted with EtOAc (3×400 mL). The combined organic layers were washedwith 1M HCl (400 mL), saturated aqueous NaHCO₃ (400 mL), and saturatedaqueous NaCl (400 mL), dried over Na₂SO₄, and concentrated to give thecrude product which was purified by chromatography to yield Compound 4(8.8 g) as a white semi-solid. LC-MS: 426.1 [M+Na⁺].

A solution of Compound 4 (8.8 g, 21.8 mmol) in EtOH (12 mL) was added toconcentrated HCl (200 mL) and heated at 100° C. and stirred overnight.The mixture was then concentrated to give the crude product which waspurified by washing with Et₂O (100 mL) to yield the title compound as asolid HCl salt (7.5 g). LC-MS: 338 [M+H⁺].

Preparation 12:(2R,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxpentanoicAcid Ethyl Ester

A solution of Compound 5 (7.5 g, 20.1 mmol) in EtOH/HCl (100 mL) washeated at 50° C. overnight. The mixture was concentrated and the crudeproduct was purified by washing with Et₂O (200 mL) to yield the titlecompound (6.5 g) as a white solid HCl salt. LC-MS: 366.1 [M+H⁺].

Preparation 13:(2R,4R)-5-(4-Bromophenyl)-2-hydroxy-4-[(1-hydroxy-1H-1,2,3-triazole-4-carbonyl)amino]pentanoicAcid Ethyl Ester

1-Hydroxy-1H-1,2,3-triazole-4-carboxylic acid was prepared by combining1-hydroxy-1H-[1,2,3]triazole-4-carboxylic acid ethyl ester (2.0 g, 13mmol), EtOH (25 mL, 430 mmol), and a pre-dissolved solution of LiOHmonohydrate (1.6 g, 38.2 mmol) and water (10 mL, 600 mmol). The mixturewas stirred at room temperature for 4 hours, then partially concentratedand acidified with HCl to cause precipitation. The solid was filteredand dried under vacuum to give 1.3 g of the desired acid.

1-Hydroxy-1H-1,2,3-triazole-4-carboxylic acid (163 mg, 1.3 mmol) wascombined with HCTU (523 mg, 1.3 mmol) and DMF, and stirred for 5 minutesat room temperature. DIPEA (661 μL, 3.8 mmol) and(2R,4R)-4-amino-5-(4-bromophenyl)-2-hydroxypentanoic acid ethyl ester(0.400 g, 1.26 mmol) were added, and the resulting mixture was stirredfor 10 minutes. The mixture was evaporated under reduced pressure andpurified (C18 column; 20-70% MeCN in water with 5% TFA) to yield thetitle compound (330 mg).

Preparation 14: 5-(4-Methoxybenzyloxy)-1-methyl-1H-pyrazole-3-carboxylicAcid

To a stirred solution of 5-hydroxy-1-methyl-1H-pyrazole-3-carboxylicacid methyl ester (200 mg, 1 mmol) in DMF (992 μL, 12.8 mmol) at 0° C.was added K₂CO₃ (195 mg, 1.4 mmol). After 10 minutes at 0° C.p-methoxybenzyl chloride (208 μL, 1.5 mmol) was added, and the resultingmixture was stirred at 60° C. for 1 hour, before allowing to cool toroom temperature. MeOH (2.6 mL, 63.2 mmol) was added, followed by LiOH(61.4 mg, 2.6 mmol) in water (2.6 mL, 142 mmol), and the reaction wasmonitored for completion. The solvent was removed in vacuo. Water wasadded to the vial and the crude product was acidified with a 1N HClaqueous solution. The solids were filtered to yield the title compoundas a white solid (350 mg).

Preparation 15:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-{[5-(4-methoxybenzyloxy)-1-methyl-1H-pyrazole-3-carbonyl]amino}pentanoicAcid

5-(4-Methoxy-benzyloxy)-1-methyl-1H-pyrazole-3-carboxylic acid (118.8mg, 453 μmol) was combined with HCTU (187.3 mg, 453 μmol) and DMF (2.0mL, 25.9 mmol). The mixture was stirred at room temperature for 15minutes. DIPEA (225 μL, 1.3 mmol) and(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (150 mg, 430 μmol) were added and the mixture was stirred at roomtemperature for 15 minutes. The solvent was removed in vacuo. EtOH (1.5mL, 25.9 mmol) was added, followed by a solution of 1 M LiOH in water(4.3 mL, 4.3 mmol). The mixture was stirred at room temperature for 1hour. The solvent was removed in vacuo and the product was purified byreverse phase chromatography to yield the title compound (171 mg).

Preparation 16: 3-(2-Fluorophenyl)-isoxazole-5-carbonyl Chloride

Oxalyl chloride (0.07 mL, 0.74 mmol) was added dropwise to a stirredsolution of 3-(2-fluorophenyl)isoxazole-5-carboxylic acid (77 mg, 370μmol) in THF (10 mL). The mixture was stirred at room temperature for 2hours, then concentrated to yield the title compound (79 mg), which wasused without further purification.

Preparation 17: 3-Methoxyisoxazole-5-carbonyl Chloride

To a solution of 3-methoxyisoxazole-5-carboxylic acid (420 mg, 3 mmol)in THF (15 mL) and DMF (1 drop) was added dropwise oxalyl chloride (650μL, 7 mmol) at 0° C. The mixture was then stirred at room temperaturefor 3 hours. The solution was concentrated in vacuo to yield the titlecompound as a light yellow oil (430 mg).

Preparation 18: (S)-2-t-Butoxycarbonylamino-3-methylbutyric AcidChloromethyl Ester

To a mixture of (S)-2-(t-butoxycarbonylamino)-3-methylbutanoic acid(28.6 g, 130 mmol) and NaHCO₃ (44 g, 520 mmol) and Bu₄NHSO₄ (4.4 g, 13mmol) in DCM (200 mL) and water (200 mL) was added chloromethylsulfochloridate (26 g, 158 mmol) at 0° C. The mixture was stirred atroom temperature for 24 hours, and then was extracted with DCM (3×150mL). The combined organic layers were washed with water (2×300 mL), andthe DCM layer was purified by flash column (petroleum ether:EA=15:1) toyield the title compound as a yellow solid (35 g). LC-MS: 266 [M+H]⁺.

Preparation 19: (S)-2-Methoxycarbonylamino-3-methylbutyric AcidChloromethyl Ester

A solution of (S)-2-t-butoxycarbonylamino-3-methylbutyric acidchloromethyl ester (35 g, 132 mmol) in DCM (200 mL) was added dropwise asolution of TFA (50 mL) in DCM (100 mL) at 0° C. The resulting mixturewas stirred at room temperature overnight and then concentrated in vacuoto yield crude Compound 1 as a yellow oil (21.8 g). LC-MS: 166 [M+H]⁺.

To a mixture of Compound 1 (21.8 g, 139 mmol) and methyl chloroformate(12 mL, 157 mmol) in THF (1 L) was added TEA (38 mL, 278 mmol) at 0° C.The resulting mixture was stirred at room temperature for 12 hours, thenconcentrated in vacuo. The residue was purified by flash column(petroleum ether:EtOAc=6:1) to yield the title compound as a yellowsolid (20.3 g). LC-MS: 224 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ0.97-1.02 (m, 6H), 2.16-2.21 (m, 1H), 3.68 (s, 1H), 4.14 (d, J=4 Hz,1H), 5.76-5.91 (m, 2H).

Preparation 20: (2R,4R)-4-Amino-5-biphenyl-4-yl-2-hydroxypentanoic AcidEthyl Ester

To a stirred solution of(S)-2-biphenyl-4-ylmethyl-5-oxopyrrolidine-1-carboxylic acid t-butylester (4.4 g, 12.4 mmol) in anhydrous THF (70 mL) was added a solutionof 1 M LiHMDS in THF (28 mL) over 15 minutes at −65° C. under nitrogen.After stirring for 3 hours at −65° C., oxodiperoxymolybdenum(pyridine)(hexamethylphosphorictriamide) (9 g, 18.6 mmol) was added. The mixturewas stirred for another 2 hours at −35° C., then saturated aqueousNa₂S₂O₃ (60 mL) was added. The organic layer was collected and washedwith saturated aqueous NH₄Cl (60 mL×3) and saturated aqueous NaCl (60mL×2), then dried over Na₂SO₄, and the solvent was removed under reducedpressure to yield the crude product which was further purified bychromatography (hexanes:EtOAc=5:1) to yield Compound 1 as a white solid(1.8 g). LC-MS: 757 [2M+Na].

To a solution of Compound 1 (1.8 g, 5.0 mmol) in anhydrous DCM (50 mL)was added DMAP (122 mg, 1 mmol) and Et₃N (1.5 g, 14.9 mmol) at 0° C.under nitrogen. After stirring for 0.5 hour at 0° C., benzyl chloride(1.0 g, 7.4 mmol) was added over 15 minutes. The mixture was stirred foran additional 2 hours at 0° C., then saturated aqueous NaHCO₃ (50 mL)was added. The organic layer was collected and washed with saturatedaqueous NaHCO₃ (50 mL×2) and saturated aqueous NaCl (50 mL×1), thendried over Na₂SO₄. The solids were filtered out and the filtrate wasconcentrated to yield the crude product which was further purified bychromatography (hexanes:EtOAc=4:1) to yield Compound 2A (471 mg) andCompound 2B (883 mg) as white solids. LC-MS: 494 [M+Na]; 965 [2M+Na].

Compound 2A: ¹H NMR (300 MHz, CDCl₃): δ (ppm)=8.02 (m, 2H), 7.57-7.25(m, 12H), 5.42 (m, 1H), 4.50 (m, 1H), 3.26-3.21 (m, 1H), 2.90 (m, 1H),2.58 (m, 1H), 2.15-2.05 (m, 1H), 1.62 (m, 9H)

Compound 2B: ¹H NMR (300 MHz, CDCl₃): δ (ppm)=8.06 (m, 2H), 7.58-7.18(m, 12H), 5.53-5.41 (m, 1H), 4.39 (m, 1H), 3.57-3.54 (m, 1H), 2.87-2.80(m, 1H), 2.48-2.44 (m, 1H), 1.98 (m, 1H), 1.63 (m, 9H).

To a stirred solution of Compound 2A (471 mg, 1 mmol) in anhydrous EtOH(10 mL) was added anhydrous K₂CO₃ (691 mg, 5 mmol) at room temperatureunder nitrogen. After stirring for 20 hours at room temperature, thesolids were filtered out. To the filtrate was added water (30 mL), DCM(30 mL) and saturated aqueous NaCl (5 mL). The aqueous layer wasseparated and extracted with DCM (30 mL×3). The combined organic layerswere washed with saturated aqueous NaCl (50 mL), dried over Na₂SO₄, andconcentrated to yield the crude product which was further purified bychromatography (hexanes:EtOAc=6:1) to yield Compound 3 as a white solid(275 mg). LC-MS: 436 [M+Na], 849 [2M+Na].

To EtOH (5 mL) was added acetyl chloride (685 mg) at −30° C. Afterstirring for 1 hour at −30° C., a solution of compound 3 (275 mg, 665μmol) in anhydrous EtOH (5 mL) was added. The mixture was heated to 25°C. and stirred for 3 hours at 25° C. After evaporation of the solvent,the residue was washed with cold anhydrous Et₂O (10 mL) to yield thetitle compound as a white solid HCl salt (207 mg). LC-MS: 314 [M+H], 649[2M+Na].

¹H NMR (300 MHz, CDCl₃): δ (ppm)=7.99 (m, 3H), 7.66-7.64 (m, 4H),7.48-7.35 (m, 5H), 6.08 (m, 1H), 4.21 (m, 1H), 4.09-4.05 (m, 2H), 3.52(m, 1H), 2.97-2.95 (m, 2H), 1.89-1.87 (m, 2H), 1.19-1.14 (m, 3H).

Preparation 21:(2R,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxypentanoicacid 5-methyl-2-oxo-[1,3]dioxol-4-ylmethyl Ester

To a suspension of(2R,4R)-4-amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxypentanoicacid (HCl salt; 10.3 g, 27.6 mmol) in THF (45 mL) was added aqueous NaOH(3.3 g, 82.8 mmol). A solution of (BOC)₂O (9.6 g, 44.2 mmol) in THF (25mL) was added dropwise at 0° C. The resulting mixture was stirred atroom temperature for 16 hours. The THF was removed and the residue wasdissolved in water (50 mL). The solution was acidified to pH=3 withaqueous HCl (2N). The resulting precipitate was filtered off, and thecake was washed with water (20 mL) and purification by chromatography(DCM:MeOH=20:1) to yield Compound 1 as a white solid (9.0 g). LC-MS: 438[M+H]⁺.

To a solution of Compound 1 (2 g, 4.6 mmol) and4-(bromomethyl)-5-methyl-1,3-dioxol-2-one (880 mg, 4.6 mmol) in DMF (20mL) was added K₂CO₃ (947 mg, 6.8 mmol) and KI (152 mg, 0.9 mmol). Afterstirring at room temperature for 2 hours, the mixture was diluted withwater (50 mL) and extracted with EtOAc (2×100 mL). The combined organiclayers were washed with saturated aqueous NaCl (2×100 mL), dried overanhydrous Na₂SO₄, filtered, and concentrated. The residue was purifiedby chromatography (DCM:EtOAc=10:1) to yield Compound 2 as a yellow foam(2.3 g). LC-MS: 572 [M+Na]⁺.

A solution of Compound 2 (2.3 g, 4.2 mmol) in HCl/dioxane (50 mL, 3.3 M)was stirred at room temperature for 2 hours. The solvent was removed,and the residue was washed with EtOAc (10 mL) to yield the titlecompound as a white solid (1.8 g). LC-MS: 450.0 [M+H]. ¹H NMR (DMSO-d₆,400 MHz) δ 1.85-1.92 (m, 2H), 2.17 (s, 3H), 2.99 (br, 2H), 3.57 (br,1H), 4.26-4.42 (m, 1H), 5.02 (s, 2H), 6.19 (d, J=5.0 Hz, 1H), 7.34-7.45(m, 3H), 7.49-7.51 (m, 1H), 7.53-7.68 (m, 3H), 7.99 (s, 3H).

Preparation 22:(2R,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxypentanoicacid 2,2,3,3,3-pentafluoropropyl Ester

Using the procedures described herein, the title compound can also beprepared.

Preparation 23:(2R,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxpentanoicAcid Butyryloxymethyl Ester

Using the procedures described herein, the title compound can also beprepared.

Example 1A:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicAcid

1-Hydroxy-1H-1,2,3-benzotriazole-6-carboxylic acid (56.6 mg, 316 μmol)and HCTU (131 mg, 316 μmol) were combined in DMF, and stirred for 5minutes at room temperature. DIPEA (83 μL, 474 μmol) and(2R,4R)-4-amino-5-(4-bromophenyl)-2-hydroxypentanoic acid ethyl ester(50 mg, 0.2 mmol) were added, and the resulting mixture was stirred for10 minutes. The mixture was evaporated under reduced pressure andpurified (C18 column. 20-70% MeCN in water with 5% TFA). The cleanfractions were lyophilized (40 mg). These solids were then combined with3-chlorophenylboronic acid (44.5 mg, 285 μmol), K₂CO₃ (66 mg, 474 μmol),EtOH (0.8 mL, 10 mmol), and water (0.2 mL, 10 mmol). SilicaCat®DPP-Pd(0.28 mmol/g loading; 57 mg, 16 μmol) was added, and the resultingmixture was microwaved at 100° C. for 10 minutes. The mixture wasfiltered and 1 M LiOH in water (1.3 mL, 1.3 mmol) was added. The mixturewas stirred for 30 minutes, evaporated under reduced pressure, andpurified by preparative HPLC to yield the title compound (20.2 mg,purity 95%). MS m/z [M+H]⁺ calc'd for C₂₄H₂₁ClN₄O₅, 481.12. found 481.2.

Example 1B:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicAcid 3,3,3-Trifluoropropyl Ester

4.0 M HCl in 1,4-dioxane (196 μL, 786 μmol) was added to a solution of(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicacid ethyl ester (50.0 mg, 98.2 μmol) in 3,3,3-trifluoropropan-1-ol (336mg, 3.0 mmol), and the resulting mixture was stirred at 70° C. for 3hours. The mixture was concentrated in vacuo to yield a white solid,which was purified by preparative HPLC (C18 column chromatography using40-90% MeCN in water with 0.05% TFA) to yield the title compound as awhite solid (18 mg, purity 100%). MS m/z [M+H]⁺ calc'd forC₂₇H₂₄ClF₃N₄O₅, 577.14. found 577.1.

Example 1C:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicAcid 2,2,2-Trifluoroethyl Ester

4.0 M HCl in 1,4-dioxane (196 μL, 786 μmol) was added to a solution of(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicacid ethyl ester (50.0 mg, 98.2 μmol) in 2,2,2-trifluoroethanol (215 μL,3.0 mmol), and the resulting mixture was stirred at 100° C. for 3 hours.The mixture was concentrated in vacuo to yield a clear colorless liquid,which was purified by preparative HPLC (C18 column chromatography using40-90% MeCN in water with 0.05% TFA) to yield the title compound as awhite solid (15.2 mg, purity 99%). MS m/z [M+H]⁺ calc'd forC₂₆H₂₂ClF₃N₄O₅, 563.12. found 563.2.

Example 1D:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicAcid 2,2,3,3,3-Pentafluoropropyl Ester

HATU (37.9 mg, 99.6 μmol) was added to a solution of1-hydroxy-1H-1,2,3-benzotriazole-6-carboxylic acid (14.9 mg, 83 μmol) inDMF (0.5 mL, 6.4 mmol) and the resulting mixture was stirred at roomtemperature for 5 minutes.(2R,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid2,2,3,3,3-pentafluoropropyl ester (30.0 mg, 66.4 μmol) was added to themixture, followed by DIPEA (35 μL, 0.2 mmol) dropwise over 5 minutes,and the mixture was stirred at room temperature for 30 minutes. Themixture was concentrated in vacuo to yield a yellow liquid, which waspurified by preparative HPLC to yield the title compound as a whitesolid (2.9 mg, purity 100%). MS m/z [M+H]⁺ calc'd for C₂₇H₂₂ClF₅N₄O₅,613.12. found 613.0.

Example 1E:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicAcid 5-Methyl-2-oxo-[1,3]dioxol-4-ylmethyl Ester

HATU (37.9 mg, 99.6 μmol) was added to a solution of1-hydroxy-1H-1,2,3-benzotriazole-6-carboxylic acid (14.9 mg, 83 μmol) inDMF (0.5 mL, 6.4 mmol) and the resulting mixture was stirred at roomtemperature for 5 minutes.(2R,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid5-methyl-2-oxo[1,3]dioxol-4-ylmethyl ester (28.7 mg, 66.4 μmol) wasadded to the mixture, followed by DIPEA (35 μL, 0.2 mmol) dropwise over5 minutes, and the mixture was stirred at room temperature for 30minutes. The mixture was concentrated in vacuo to yield a yellow liquid,which was purified by preparative HPLC to yield the title compound as awhite solid (37.8 mg, purity 98.5%). MS m/z [M+H]⁺ calc'd forC₂₉H₂₅ClN₄O₈, 593.14. found 593.0.

Example 1F:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicAcid Butyryloxymethyl Ester

HATU (37.9 mg, 99.6 μmol) was added to a solution of1-hydroxy-1H-1,2,3-benzotriazole-6-carboxylic acid (14.9 mg, 83 μmol) inDMF (0.5 mL, 6.4 mmol) and the resulting mixture was stirred at roomtemperature for 5 minutes.(2R,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acidbutyryloxymethyl ester (27.9 mg, 66.4 μmol) was added to the mixture,followed by DIPEA (35 μL, 0.2 mmol) dropwise over 5 minutes, and themixture was stirred at room temperature for 30 minutes. The mixture wasconcentrated in vacuo to yield a yellow liquid, which was purified bypreparative HPLC to yield the title compound as a white solid (5.2 mg,purity 98.2%). MS m/z [M+H]⁺ calc'd for C₂₉H₂₉ClN₄O₇, 581.17. found581.2.

Example 1G:(2R,4R)-4-[(3-Acetoxymethoxy-3H-benzotriazole-5-carbonyl)amino]-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicAcid

4.0 M HCl in 1,4-dioxane (270 mL, 1.1 mmol) was added to a solution of(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicacid (130 mg, 270 μmol) in benzyl alcohol (559 μL, 5.4 mmol), andstirred at 60° C. for 1.5 hours. The mixture was then purified (C18column chromatography, 55 g column, using 30-90% MeCN in water with0.05% TFA) to yield an off-white solid. Bromomethyl acetate (45.5 mg,297 μmol) was added to a solution of this off-white solid and K₂CO₃(82.2 mg, 595 μmol) in DMF (3.1 mL, 40.0 mmol) and the resulting mixturewas stirred at room temperature for 1 hour. The mixture was thenpurified (column chromatography, small column, using 40-90% MeCN inwater with 0.05% TFA) to yield a clear colorless viscous liquid. 10%Pd/C, 50% wet (0.45 mmol/g loading; 30.0 mg, 13.5 μmol) was added to asolution of this clear colorless viscous liquid in MeOH (4.1 mL, 102mmol) and the resulting mixture was stirred under hydrogen for 30minutes. The mixture was filtered and the filtrate was concentrated invacuo to yield a clear colorless liquid. The crude liquid was purified(C18 column chromatography, 55 g column, using 30-85% MeCN in water with0.05% TFA) to yield the title compound (14.0 mg) as a white solid. MSm/z [M+H]⁺ calc'd for C₂₇H₂₅ClN₄O₇, 553.14. found 553.1.

Example 1H:(2R,4R)-4-[(3-Acetoxymethoxy-3H-benzotriazole-5-carbonyl)amino]-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicAcid Acetoxymethyl Ester

Et₃N (7.0 μL, 50 μmol) was added to a solution of[(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicacid (20.0 mg, 42 μmol) and bromomethyl acetate (6.52 μL, 67 μmol) inacetone (1.3 mL, 17.2 mmol), and the resulting mixture was stirred in asealed vial at 65° C. for 5 hours. 1:1 TFA/DCM (0.50 mL, 3.1 mmol) wasadded, and the mixture was stirred at room temperature for 30 minutesand concentrated to yield a clear yellow liquid. The crude liquid waspurified by preparative HPLC (C18 column chromatography, small column,using 30-90% MeCN in water with 0.05% TFA) to yield the title compound(5.2 mg, purity 100%). MS m/z [M+H]⁺ calc'd for C₃₀H₂₉ClN₄O₉, 625.16.found 625.1.

Example 1I:(2R,4R)-4-[(3-Benzyloxy-3H-benzotriazole-5-carbonyl)amino]-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicAcid

HATU (246 mg, 647 μmol) was added to a solution of1-hydroxy-1H-1,2,3-benzotriazole-6-carboxylic acid (116 mg, 647 μmol) inDMF (2.0 mL, 25.8 mmol) and the resulting mixture was stirred at roomtemperature for 10 minutes. DIPEA (225 μL, 1.3 mmol) was added to themixture, followed by(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (150 mg, 431 μmol) and the mixture was stirred at room temperaturefor 1 hour. 3.0 M LiOH in water (1.7 mL, 5.2 mmol) was added to themixture and stirred at room temperature for 30 minutes. The mixture wasthen purified (C18 column chromatography, 55 g column, using 40-90% MeCNin water with 0.05% TFA) to yield a white solid. Benzyl bromide (41 μL,345 μmol) was added to a solution of this solid in acetone (3.0 mL, 40.8mmol). Et₃N (54 μL, 388 μmol) was added and the resulting mixture wasstirred at 50° C. for 2 hours. The mixture was concentrated in vacuo andthe resulting crude clear yellow liquid was purified (C18 columnchromatography, 55 g column, using 40-90% MeCN in water with 0.05% TFA)to yield the title compound (201 mg, purity 100%) as a white solid. MSm/z [M+H]⁺ calc'd for C₃₁H₂₇ClN₄O₅, 571.17. found 571.

Example 1J:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicAcid Benzyl Ester

4.0 M HCl in 1,4-dioxane (270 μL, 1.0 mmol) was added to a solution of(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicacid (130 mg, 270 μmol) in benzyl alcohol (559 μL, 5.4 mmol), and theresulting mixture was stirred at 60° C. for 15 minutes. The mixture waspurified by preparative HPLC (C18 column chromatography using 30-90%MeCN in water with 0.05% TFA) to yield the title compound as anoff-white solid (16.0 mg, purity 99.2%). MS m/z [M+H]⁺ calc'd forC₃₁H₂₇ClN₄O₅, 571.17. found 571.1.

Example 1K:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicAcid Acetoxymethyl Ester

Bromomethyl acetate (6.7 μL, 68.3 μmol) and Et₂O (11.7 μL, 84 μmol) wereadded to a solution of(2R,4R)-4-[(3-benzyloxy-3H-benzotriazole-5-carbonyl)amino]-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicacid (30.0 mg, 52.5 μmol) in acetone (1.0 mL, 13.6 mmol) and theresulting mixture was stirred at 45° C. for 1 hour. A drop of AcOH wasadded to neutralize the reaction and the mixture was concentrated toyield a clear yellow liquid. The crude liquid was purified (C18 columnchromatography, 55 g column, using 40-90% MeCN in water with 0.05% TFA)to yield a white solid. The solid was dissolved in THF (1.0 mL, 12.3mmol) and 10% Pd/C, 50% wet (0.45 mmol/g loading; 5.8 mg, 2.6 μmol) wasadded to the solution, which was stirred under hydrogen for 1 hour. Themixture was filtered and the filtrate was concentrated to yield a clearcolorless liquid. The crude liquid was purified (preparative scale C18column chromatography, small column, using 30-90% MeCN in water with0.05% TFA) to yield the title compound as a white solid (10.0 mg, purity99%). MS m/z [M+H]⁺ calc'd for C₂₇H₂₅ClN₄O₇, 553.14. found 553.1.

Example 1L:(2R,4R)-4-{[3-((R)-2-Amino-3-methylbutyryloxymethoxy)-3H-benzotriazole-5-carbonyl]amino}-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-pentanoicAcid

Et₃N (7.0 μL, 50 μmol) was added to a solution of[(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicacid (20.0 mg, 42 μmol) and (S)-2-t-butoxycarbonylamino-3-methylbutyricacid chloromethyl ester (17.7 mg, 67 μmol) in acetone (1.3 mL, 17.2mmol), and the resulting mixture was stirred in a sealed vial at 65° C.for 5 hours. 1:1 TFA/DCM (0.50 mL, 3.1 mmol) was added, and the mixturewas stirred at room temperature for 30 minutes and concentrated to yielda clear yellow liquid. The crude liquid was purified by preparative HPLC(C18 column chromatography, small column, using 30-90% MeCN in waterwith 0.05% TFA) to yield to yield the title compound as a white solidHCl salt (6.2 mg, purity 96%). MS m/z [M+H]⁺ calc'd for C₃₀H₃₂ClN₅O₇,610.20. found 610.1.

Example 1M:(2R,4R)-4-[(3-Butyryloxymethoxy-3H-benzotriazole-5-carbonylamino]-5-(3′-chlorobiphenyl-4-yl)-2-hydroxpentanoicAcid

Et₃N (7.0 μL, 50 μmol) was added to a solution of[(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicacid (20.0 mg, 42 μmol) and chloromethyl butyrate (8.3 μL, 67 μmol) inacetone (1.3 mL, 17.2 mmol), and the resulting mixture was stirred in asealed vial at 65° C. for 5 hours. The mixture was then concentrated toyield a yellow liquid. The crude liquid was purified by preparative HPLC(C18 column chromatography, small column, using 30-90% MeCN in waterwith 0.05% TFA) to yield the title compound as a white solid (3.3 mg,purity 94%). MS m/z [M+H]⁺ calc'd for C₂₉H₂₉ClN₄O₇, 581.17. found 581.1.

Example 1N:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicAcid (R)-2-Methoxycarbonylamino-3-methylbutyryloxymethyl Ester

(S)-2-t-Butoxycarbonylamino-3-methyl-butyric acid chloromethyl ester(18.1 mg, 68.3 μmol) and Et₃N (11.7 μL, 84 μmol) were added to asolution of(2R,4R)-4-[(3-benzyloxy-3H-benzotriazole-5-carbonyl)amino]-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicacid (30.0 mg, 52.5 μmol) in acetone (1.00 mL, 13.6 mmol) and stirred at60° C. for 3 hours. The mixture was concentrated to yield a yellowliquid, which was combined with 1:1 TFA/DCM (1.0 mL, 6.2 mmol) andstirred at room temperature for 1 hour. The mixture was concentrated invacuo and the resulting crude liquid was purified (C18 columnchromatography, 55 g column, using 30-90% MeCN in water with 0.05% TFA)to yield a white solid. The solid was dissolved in DCM (0.5 mL, 7.8mmol) at 0° C. Methyl chloroformate (6.1 μL, 78.8 μmol) was addedfollowed by Et₃N (18.3 μL, 131 μmol) and the resulting mixture wasstirred at room temperature for 30 minutes. A drop of AcOH was added toneutralize the reaction and the mixture was concentrated to yield aclear yellow liquid. The crude liquid was combined with 10% Pd/C, 50%wet (0.45 mmol/g loading; 5.8 mg, 2.6 μmol) and stirred under hydrogenfor 1 hour. The mixture was filtered and the filtrate was concentratedto yield a clear yellow liquid. The crude liquid was purified(preparative scale C18 column chromatography, small column, using 30-90%MeCN in water with 0.05% TFA) to yield the title compound as a whitesolid TFA salt (7.5 mg, 99% purity). MS m/z [M+H]⁺ calc'd forC₃₂H₃₄ClN₅O₉, 668.20. found 668.1.

Example 1O:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-{[3-((S)-2-methoxycarbonylamino-3-methylbutyryloxymethoxy)-3H-benzotriazole-5-carbonyl]amino}pentanoicAcid

Et₃N (7.0 μL, 50 μmol) was added to a solution of[(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicacid (20.0 mg, 42 μmol) and chloromethyl butyrate (8.3 μL, 67 μmol) inacetone (1.3 mL, 17.2 mmol), and the resulting mixture was stirred in asealed vial at 65° C. for 5 hours. The mixture was then concentrated toyield a yellow liquid. The crude liquid was purified by preparative HPLC(C18 column chromatography, small column, using 30-90% MeCN in waterwith 0.05% TFA) to yield the title compound as a white solid (3.3 mg,purity 94%). MS m/z [M+H]⁺ calc'd for C₂₉H₂₉ClN₄O₇, 581.17. found 581.1.

Example 1P:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-hydroxy-3H-benzotriazole-5-carbonyl)amino]pentanoicAcid (R)-2-amino-3-methylbutyryloxymethyl Ester

(S)-2-t-Butoxycarbonylamino-3-methyl-butyric acid chloromethyl ester(18.1 mg, 68.3 μmol) and Et₃N (11.7 μL, 84 μmol) were added to asolution of(2R,4R)-4-[(3-benzyloxy-3H-benzotriazole-5-carbonyl)amino]-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicacid (30.0 mg, 52.5 μmol) in acetone (1.00 mL, 13.6 mmol) and stirred at60° C. for 3 hours. The mixture was concentrated to yield a yellowliquid, which was combined with 1:1 TFA/DCM (1.0 mL, 6.2 mmol) andstirred at room temperature for 1 hour. The mixture was concentrated invacuo and the resulting crude liquid was purified (C18 columnchromatography, 55 g column, using 30-90% MeCN in water with 0.05% TFA)to yield a white solid. The solid was dissolved in THF (1.0 mL) and 10%Pd/C, 50% wet (0.45 mmol/g loading; 5.8 mg, 2.6 μmol) was added to thesolution, which was stirred under hydrogen for 1 hour. The mixture wasfiltered and the filtrate was concentrated to yield a clear yellowliquid. The crude liquid was purified (preparative scale C18 columnchromatography, small column, using 30-90% MeCN in water with 0.05% TFA)to yield the title compound as a white solid TFA salt (6.5 mg, purity98%). MS m/z [M+H]⁺ calc'd for C₃₀H₃₂ClN₅O₇, 610.20. found 610.1.

Example 2A:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-hydroxy-1-methyl-1H-pyrazole-3-carbonyl)amino]pentanoicAcid

5-Hydroxy-1-methyl-1H-pyrazole-3-carboxylic acid (19.6 mg, 138 μmol) wascombined with HCTU (56.9 mg, 138 μmol) in DMF. The mixture was stirredfor 10 minutes at room temperature before adding DIPEA (72 μL, 413 μmol)and (2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acidethyl ester (50 mg, 0.1 mmol). The resulting mixture was stirredovernight at room temperature. EtOH (402 μL, 6.9 mmol) and 1 M LiOH inwater (1.1 mL, 1.1 mmol) was added and the mixture was stirred at roomtemperature for 1 hour. The mixture was evaporated under reducedpressure and purified by preparative HPLC to yield the title compound(6.4 mg). MS m/z [M+H]⁺ calc'd for C₂₂H₂₂ClN₃O₅, 444.12. found 444.4.

Example 2B:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-hydroxy-1-methyl-1H-pyrazole-3-carbonyl)amino]pentanoicAcid 5-Methyl-2-oxo-[1,3]dioxol-4-ylmethyl Ester

5-Hydroxy-1-methyl-1H-pyrazole-3-carboxylic acid (5.6 mg, 39 μmol) andHCTU (16.2 mg, 39 μmol) were combined in DMF (166 μL, 2.1 mmol) andstirred at room temperature for 15 minutes. DIPEA (19 μL, 0.1 mmol) and(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid5-methyl-2-oxo[1,3]dioxol-4-ylmethyl ester (15.4 mg, 36 μmol) wereadded, and the resulting mixture was stirred at room temperature for 30minutes. The solvent was removed in vacuo and the crude product waspurified by preparative HPLC to yield the title compound (7 mg). MS m/z[M+H]⁺ calc'd for C₂₇H₂₆ClN₃O₈, 556.14. found 556.1.

Example 2C:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-hydroxy-1-methyl-1H-pyrazole-3-carbonyl)amino]pentanoicAcid Ethyl Ester

5-Hydroxy-1-methyl-1h-pyrazole-3-carboxylic acid (122 mg, 862 μmol) andHATU (361 mg, 949 μmol) were combined in DMF (1.0 mL, 13 mmol) andstirred for 10 minutes.(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (30.0 mg, 86 μmol) and DIPEA (165 μL, 949 μmol) were added, andthe resulting mixture was stirred at room temperature overnight. Themixture was then concentrated under reduced pressure and the residue wasdissolved in 50% AcOH-water (1.5 mL), filtered, purified by reversephase preparative HPLC, and lyophilized to yield the title compound(11.1 mg). MS m/z [M+H]⁺ calc'd for C₂₄H₂₆ClN₃O₅, 472.16. found 472.2.

Example 2D:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-hydroxy-1-methyl-1H-pyrazole-3-carbonyl)amino]pentanoicAcid 2,2,3,3,3-Pentafluoropropyl Ester

(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-{[5-(4-methoxybenzyloxy)-1-methyl-1H-pyrazole-3-carbonyl]amino}pentanoicacid (28.0 mg, 49.6 μmol) was combined with HOBt (40.2 mg, 298 μmol) andEDC (53 μL, 0.3 mmol) in DCM (0.4 mL, 6 mmol). The resulting solutionwas stirred for 15 minutes before the addition of2,2,3,3,3-pentafluoro-1-propanol (39.6 μL, 397 μmol). The mixture wasstirred at room temperature and the reaction monitored for completion.After 4 hours, the mixture was concentrated by rotary evaporation andthe reside was purified (reverse phase column).

The residue was dissolved in EtOAc (842 μL, 8.6 mmol) and 10% Pd/C, 50%wet (0.45 mmol/g loading; 31.9 mg, 14 μmol) was added. The resultingmixture was stirred under hydrogen for 1 hour. The solvent was removedin vacuo and the residue was purified by preparative HPLC to yield thetitle compound (4.9 mg, purity 100%). MS m/z [M+H]⁺ calc'd forC₂₅H₂₃ClF₅N₃O₅, 576.12. found 576.1.

Example 2E:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-hydroxy-1-methyl-1H-pyrazole-3-carbonyl)amino]pentanoicAcid 1-Trifluoromethylpropyl Ester

(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-{[5-(4-methoxybenzyloxy)-1-methyl-1H-pyrazole-3-carbonyl]amino}pentanoicacid (28.0 mg, 49.6 μmol) was combined with HOBt (40.2 mg, 298 μmol) andEDC (53 μL, 0.3 mmol) in DCM (0.4 mL, 6 mmol). The resulting solutionwas stirred for 15 minutes before the addition of1,1,1-trifluoro-2-butanol (50.9 mg, 397 μmol). The mixture was stirredat room temperature and the reaction monitored for completion. After 4hours, the mixture was concentrated by rotary evaporation and the residewas purified (reverse phase column).

The residue was dissolved in EtOAc (842 μL, 8.6 mmol), and 10% Pd/C, 50%wet (0.45 mmol/g loading; 31.9 mg, 14 μmol) was added. The resultingmixture was stirred under hydrogen for 1 hour. The solvent was removedin vacuo and the residue was purified by preparative HPLC to yield thetitle compound (1.3 mg, purity 99%). MS m/z [M+H]⁺ calc'd forC₂₆H₂₇ClF₃N₃O₅, 554.16. found 554.1.

Example 2F:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-hydroxy-1-methyl-1H-pyrazole-3-carbonyl)amino]pentanoicAcid 2,2,3,3,3-Pentafluoro-1-Methylpropyl Ester

(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-{[5-(4-methoxybenzyloxy)-1-methyl-1H-pyrazole-3-carbonyl]amino}pentanoicacid (28.0 mg, 49.6 μmol) was combined with HOBt (40.2 mg, 298 μmol) andEDC (53 μL, 0.3 mmol) in DCM (0.4 mL, 6 mmol). The resulting solutionwas stirred for 15 minutes before the addition of3,3,4,4,4-pentafluoro-2-butanol (65.2 mg, 397 μmol). The mixture wasstirred at room temperature and the reaction monitored for completion.After 4 hours, the mixture was concentrated by rotary evaporation andthe reside was purified (reverse phase column).

The residue was dissolved in EtOAc (842 μL, 8.6 mmol), and 10% Pd/C, 50%wet (0.45 mmol/g loading; 31.9 mg, 14 μmol) was added. The resultingmixture was stirred under hydrogen for 1 hour. The solvent was removedin vacuo and the residue was purified by preparative HPLC to yield thetitle compound (1.8 mg, purity 98%). MS m/z [M+H]⁺ calc'd forC₂₆H₂₅ClF₅N₃O₅, 590.14. found 590.1.

Example 2G:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-hydroxy-1-methyl-1H-pyrazole-3-carbonyl)amino]pentanoicAcid Benzyl Ester

A mixture of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (150.0 mg, 431 μmol), benzyl alcohol (446.2 μL, 4.3 mmol), and 4.0M HCl in 1,4-dioxane (431 μL, 1.7 mmol) was stirred at room temperatureovernight, then at 50° C. for 1 hour. The mixture was then concentratedunder reduced pressure. 5-Hydroxy-1-methyl-1H-pyrazole-3-carboxylic acid(30.6 mg, 216 μmol) and HATU (98.4 mg, 259 μmol) were stirred in DMF(0.5 mL, 5 mmol) for 10 minutes, then added to the concentrated mixture,along with DIPEA (113 μL, 647 μmol). The resulting mixture was stirredat room temperature for 2 hours and concentrated under reduced pressure.The residue was dissolved in 50% AcOH-water (5 mL), filtered, andpurified by reverse phase preparative HPLC to yield the title compound(81.4 mg).

Example 2H:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-hydroxy-1-methyl-1H-pyrazole-3-carbonyl)amino]pentanoicAcid Butyryloxymethyl Ester

To a solution of 5-hydroxy-1-methyl-1H-pyrazole-3-carboxylic acid methylester (3 g, 19.2 mmol) in DMF (15 mL) was added K₂CO₃ (2.7 g, 19.2 mmol)at room temperature. After 10 min, allyl bromide (2.3 g, 19.2 mmol) wasadded, and the resulting mixture was stirred at room temperature for 2hours. Water (150 mL) was added, the mixture was extracted with EtOAc(3×50 mL), and the combined organic layers were washed with saturatedaqueous NaCl (50 mL) and dried over anhydrous Na₂SO₄. The solution wasthen evaporated and the residue was purified by silica gelchromatography (silica gel: 200-300 mesh, eluted with PE:EtOAc=10:1 to5:1 to 1:1) to yield Compound 1 as a white solid (3 g). LC-MS: 197[M+H]⁺.

To a solution of Compound 1 (3 g, 15 mmol) in THF (30 mL) was added asolution of LiOH (550 mg, 23 mmol) in water (15 mL). The mixture wasstirred at room temperature for 1 hour, then concentrated in vacuo.Water was added (20 mL) to the residue and the mixture was washed withEtOAc (20 mL). The aqueous layer was acidified with 1N aqueous HCl topH=3 and extracted with EtOAc (3×30 mL). The combined organic layerswere washed with saturated aqueous NaCl (30 mL), and dried overanhydrous Na₂SO₄. The solution was then evaporated to yield Compound 2as a white solid (2.3 g). LC-MS: 183 [M+H]⁺.

To a solution of(2R,4R)-4-amino-2-hydroxy-5-(3′-methylbiphenyl-4-yl)pentanoic acid ethylester (13 g, 7.1 mmol) and Compound 2 (2.7 g, 7.1 mmol) in DMF (30 mL)were added PyBOP (3.7 g, 7.1 mmol) and DIPEA (1.8 g, 14.2 mmol) at 0° C.The mixture was stirred at room temperature for 2 hours. Water was added(200 mL), the mixture was extracted with EtOAc (3×100 mL), and thecombined organic layers were washed with saturated aqueous NaCl (100 mL)and dried over anhydrous Na₂SO₄. The mixture was concentrated and theresidue was purified by silica gel chromatography (silica gel: 200-300mesh; eluted with PE:EtOAc=10:1 to 5:1 to 1:1) to yield Compound 3 as alight yellow solid (2.4 g). LC-MS: 512 [M+H]⁺.

To a solution of Compound 3 (2.4 g, 4.7 mmol) in THF (20 mL) and water(10 mL) was added LiOH (169 mg, 7 mmol) at room temperature. Theresulting mixture was stirred at room temperature for 1 hour. Themixture was concentrated, water was added (50 mL) and the mixture waswashed with EtOAc (2×20 mL). The aqueous layer was acidified with 1Naqueous HCl to pH=3 and extracted with EtOAc (3×50 mL). The combinedorganic layers were washed with saturated aqueous NaCl (50 mL) and driedover anhydrous Na₂SO₄. The solution was then evaporated to yieldCompound 4 as a yellow solid (2 g). LC-MS: 484 [M+H]⁺.

A suspension of Compound 4 (400 mg, 830 μmol), NaI (248 mg, 1.7 mmol)and lutidine (173 mg, 1.7 mmol) in chloromethyl butyrate (2 mL) wasstirred at 50° C. for 5 hours. After cooling to room temperature, themixture was diluted with water (20 mL) and extracted with EtOAc (2×20mL). The combined organic layers were washed with saturated aqueous NaCl(40 mL), dried over anhydrous Na₂SO₄, concentrated, and purified bysilica gel chromatography (silica gel: 200-300 mesh; elute with PE:EtOAcfrom 5:1 to 2:1) to yield Compound 5 as a light yellow solid (200 mg).LC-MS: 584 [M+H]⁺.

To a solution of Compound 5 (140 mg, 240 μmol) in DCM (5 mL) was addedPd(PPh₃)₄ (83 mg, 70 μmol), Et₃SiH (83 mg, 720 μmol) and AcOH (43 mg,720 μmol). The mixture was stirred at room temperature for 4 hours andconcentrated. The residue was purified by preparative HPLC[Daisogel-C18, 150×21.2 mm, 5μ; MeCN—H₂O (0.1% TFA) from 60% to 80%] toyield the title compound as a yellow oil (20 mg). LC-MS: 544 [M+H]⁺.¹H-NMR (CD₃OD, 400 Hz): δ 0.93 (t, 3H), 1.62 (q, 2H), 2.06-2.13 (m, 2H),2.31 (t, 2H), 2.97-2.99 (m, 2H), 3.65 (s, 3H), 4.32 (m, 1H), 4.51 (m,1H), 5.75 (dd, 2H), 7.35-7.41 (m, 4H), 7.53-7.60 (m, 4H).

Example 2I:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-hydroxy-1-methyl-1H-pyrazole-3-carbonyl)amino]pentanoicAcid (S)-2-Amino-3-methylbutyryloxymethyl Ester

A suspension of(2R,4R)-4-[(5-allyloxy-1-methyl-1H-pyrazole-3-carbonyl)amino]-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-pentanoicacid (400 mg, 830 μmol), NaI (248 mg, 1.7 mmol) and lutidine (173 mg,1.7 mmol) in (S)-2-t-butoxycarbonylamino-3-methylbutyric acidchloromethyl ester (2 mL) was stirred at 50° C. for 2 hours. Aftercooling to room temperature, the mixture was diluted with water (20 mL)and extracted with EtOAc (2×20 mL). The combined organic layers werewashed with saturated aqueous NaCl (40 mL), dried over anhydrous Na₂SO₄,concentrated, and purified by silica gel chromatography (silica gel:200-300 mesh; elute with PE:EtOAc from 5:1 to 2:1) to yield Compound 1as a light yellow solid (290 mg). LC-MS: 713 [M+H]⁺.

To a solution of Compound 1 (290 mg, 0.4 mmol) in THF (5 mL) was addedPd(PPh₃)₄ (70 mg, 60 μmol) and 1,3-dimethylbarbituric acid (312 mg, 2mmol). The mixture was stirred at room temperature for 2 hours andconcentrated. The residue was dissolved in EtOAc (30 mL), washed with 5%NaHCO₃ (2×15 mL), dried over anhydrous Na₂SO₄, and concentrated to yieldcrude Compound 2 as a brown solid (220 mg). LC-MS: 673 [M+H]⁺.

To a 1 M solution of HCl (g) in dioxane (5 mL) was added Compound 2 (220mg, 330 μmol). The mixture was stirred at room temperature for 1 hourand concentrated. The residue was purified by preparative HPLC[Daisogel-C18, 150×21.2 mm, 5μ; MeCN—H₂O (0.1% TFA) from 35% to 45%] toyield the title compound as a yellow solid (100 mg). LC-MS: 573 [M+H]⁺.¹H-NMR (DMSO-d6, 400 Hz): δ 0.97 (d, 6H), 1.82-1.85 (m, 1H), 1.98-2.01(m, 1H), 2.16-2.19 (m, 1H), 2.85-2.88 (m, 2H), 3.58 (s, 3H), 3.99-4.01(m, 1H), 4.18-4.20 (m, 1H), 4.23-4.25 (m, 1H), 5.84 (dd, 2H), 7.28-7.47(m, 4H), 7.60-7.69 (m, 4H), 8.01 (s, 1H), 8.55 (br, 2H).

Example 3A:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-hydroxy-1-pyridin-2-yl-1H-pyrazole-3-carbonyl)amino]pentanoicAcid

5-Hydroxy-1-pyridin-2-yl-1H-pyrazole-3-carboxylic acid (59.0 mg, 287μmol) and HCTU (131 mg, 316 μmol) were combined in DMF (1.3 mL, 17.2mmol), and stirred at room temperature for 15 minutes.(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid5-methyl-2-oxo[1,3]dioxol-4-ylmethyl ester (100 mg, 0.3 mmol) and DIPEA(150 μL, 862 μmol) were added, and the resulting mixture was stirred atroom temperature for 15 minutes. The reaction was evaporated underreduced pressure. The residue was dissolved in EtOH and a solution of 1M LiOH in water (1.4 mL, 1.4 mmol) was added. The resulting mixture wasstirred at room temperature for 1 hour, then evaporated under reducedpressure. The residue was purified by preparative HPLC to yield thetitle compound (85 mg). MS m/z [M+H]⁺ calc'd for C₂₆H₂₃ClN₄O₅, 507.14.found 507.1.

Example 3B:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-hydroxy-1-pyridin-2-yl-1H-pyrazole-3-carbonyl)amino]pentanoicAcid 5-Methyl-2-oxo-[1,3]dioxol-4-ylmethyl Ester

5-Hydroxy-1-pyridin-2-yl-1H-pyrazole-3-carboxylic acid (8.1 mg, 39 μmol)and HCTU (16.2 mg, 39 μmol) were combined in DMF (166 μL, 2.1 mmol) andstirred at room temperature for 15 minutes. DIPEA (19 μL, 0.1 mmol) and(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid5-methyl-2-oxo[1,3]dioxol-4-ylmethyl ester (15.4 mg, 36 μmol) wereadded, and the resulting mixture was stirred at room temperature for 30minutes. The solvent was removed in vacuo and the crude product waspurified by preparative HPLC to yield the title compound (18 mg). MS m/z[M+H]⁺ calc'd for C₃₁H₂₇ClN₄O₈, 619.15. found 619.1.

Example 3C:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-hydroxy-1-pyridin-2-yl-1H-pyrazole-3-carbonyl)amino]pentanoicAcid Ethyl Ester

5-Hydroxy-1-pyridin-2-yl-1H-pyrazole-3-carboxylic acid (23.6 mg, 115μmol) and HATU (52.5 mg, 138 μmol) were stirred in N,N-dimethylacetamide(1.0 mL, 11 mmol) for 10 minutes.(2R,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (40.0 mg, 115 μmol) and DIPEA (60.1 μL, 345 μmol) were added, andresulting mixture was stirred at room temperature for 2 hours. Themixture was then concentrated under reduced pressure, the residue wasdissolved in EtOAc (20 mL), and the organic layer was washed with water(2×5 mL), dried over MgSO₄, and concentrated. One half of the residuewas dissolved in 50% AcOH-water (1.5 mL), filtered, and purified byreverse phase preparative HPLC to yield the title compound as a TFA salt(6.1 mg). MS m/z [M+H]⁺ calc'd for C₂₈H₂₇ClN₄O₅, 535.17. found 535.4.

Example 3D:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-hydroxy-1-pyridin-2-yl-1H-pyrazole-3-carbonyl)amino]pentanoicAcid Isobutyl Ester

5-Hydroxy-1-pyridin-2-yl-1H-pyrazole-3-carboxylic acid (23.6 mg, 115μmol) and HATU (52.5 mg, 138 μmol) were stirred in N,N-dimethylacetamide(1.0 mL, 11 mmol) for 10 minutes.(2R,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (40.0 mg, 115 μmol) and DIPEA (60.1 μL, 345 μmol) were added, andresulting mixture was stirred at room temperature for 2 hours. Themixture was then concentrated under reduced pressure, the residue wasdissolved in EtOAc (20 mL), and the organic layer was washed with water(2×5 mL), dried over MgSO₄, and concentrated. One half of the residuewas combined with isobutyl alcohol (0.5 mL, 5 mmol), and 4.0 M HCl in1,4-dioxane (115 μL, 460 μmol). The mixture was stirred at roomtemperature overnight, concentrated under reduced pressure, the residuedissolved in 50% AcOH-water (1.5 ml), filtered, and purification byreverse phase preparative HPLC to yield the title compound as a TFA salt(9.1 mg). MS m/z [M+H]⁺ calc'd for C₃₀H₃₁ClN₄O₅, 563.20. found 563.4.

Example 3E:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-hydroxy-1-pyridin-2-yl-1H-pyrazole-3-carbonyl)amino]pentanoicAcid Benzyl Ester

A mixture of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (150.0 mg, 431 μmol), benzyl alcohol (446.2 μL, 4.3 mmol), and 4.0M HCl in 1,4-dioxane (431 μL, 1.7 mmol) was stirred at room temperatureovernight, then at 50° C. for 1 hour. The mixture was then concentratedunder reduced pressure.5-Hydroxy-1-pyridin-2-yl-1H-pyrazole-3-carboxylic acid (44.2 mg, 216μmol) and HATU (98.4 mg, 259 μmol) were stirred in DMF (0.5 mL, 5 mmol)for 10 minutes, then added to the concentrated mixture, along with DIPEA(113 μL, 647 μmol). The resulting mixture was stirred at roomtemperature for 2 hours and concentrated under reduced pressure. Theresidue was dissolved in 50% AcOH-water (5 mL), filtered, and purifiedby reverse phase preparative HPLC to yield the title compound (19.5 mg).

Example 3F:(2R,4R)-2-((S)-2-Amino-3-methylbutyryloxy)-5-(3′-chlorobiphenyl-4-yl)-4-[(5-hydroxy-1-pyridin-2-yl-1H-pyrazole-3-carbonyl)amino]pentanoicAcid

Using the procedures described herein, the title compound can also beprepared.

Example 3G:(2R,4R)-4-{[5-((S)-2-Amino-3-methylbutyryloxymethoxy)-1-pyridin-2-yl-1H-pyrazole-3-carbonyl]amino}-5-(3′-chloro-biphenyl-4-yl)-2-hydroxypentanoicAcid

Using the procedures described herein, the title compound can also beprepared.

Example 3H:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-hydroxy-1-pyridin-2-yl-1H-pyrazole-3-carbonyl)amino]pentanoicAcid (S)-2-methoxycarbonylamino-3-methylbutyryloxymethyl Ester

To a solution of(2R,4R)-4-[(5-allyloxy-1-pyridin-2-yl-1H-pyrazole-3-carbonyl)-amino]-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicacid (220 mg, 0.4 mmol) and ((S)-2-methoxycarbonylamino-3-methylbutyricacid chloromethyl ester (180 mg, 0.8 mmol) in DMF (2 mL) was added 2.6lutidine 2,6-lutidine (130 mg, 1.2 mmol) and NaI (60 mg, 0.4 mmol).After stirring at room temperature for 24 hours, the mixture was dilutedwith water (20 mL) and extracted with EtOAc (2×20 mL). The combinedorganic layers were washed with saturated aqueous NaCl(2×70 mL), driedover anhydrous Na₂SO₄, filtered, concentrated and purified bypreparative TLC (PE:EtOAc=1:1) to yield Compound 1 as a yellow solid(160 mg). LC-MS: 734 [M+H]⁺.

A mixture of Compound 1 (140 mg, 190 μmol), Et₃SiH (88 mg, 760 μmol),Pd(PPh₃)₄ (22 mg, 20 μmol), and AcOH (22 mg, 0.38 mmol) in DCM (2 mL)was stirred at room temperature for 4 hours. The solvent was removed andthe residue was purified by preparative HPLC (Gemini-C18 150×21.2 mm,5μ; MeCN—H₂O (0.1% TFA); from 50% to 70%) to yield the title compound asa white solid (15 mg). LC-MS: 694 [M+H]⁺. ¹H NMR (400 MHz, CD₃OD) δ 8.47(d, J=5.0 Hz, 1H), 8.06 (d, J=8.5 Hz, 2H), 7.55 (dd, J=19.3, 11.4 Hz,4H), 7.37 (dt, J=28.2, 6.2 Hz, 5H), 5.85 (d, J=5.7 Hz, 1H), 5.75 (d,J=5.7 Hz, 1H), 4.58 (m, 1H), 4.36 (t, J=5.8 Hz, 1H), 4.04 (d, J=5.9 Hz,1H), 3.65 (s, 3H), 3.04 (d, J=6.8 Hz, 2H), 2.20 (dd, J=12.5, 7.1 Hz,1H), 2.08 (m, 2H), 0.90 (dd, J=10.8, 6.9 Hz, 6H).

Example 4A:(2R,4R)-4-[(5-Acetyl-1H-pyrazole-3-carbonyl)amino]-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxypentanoicAcid Ethyl Ester

To a stirred solution of 3-acetyl-1H-pyrazole-5-carboxylic acid (50.9mg, 330 μmol), HATU (125 mg, 330 μmol), and DIPEA (104 μL, 0.6 mmol) inDMF (0.6 mL, 7.7 mmol), was added(2R,4R)-4-amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxypentanoicacid ethyl ester (110 mg, 300 μmol). The resulting mixture was stirredat room temperature until the reaction was complete. The mixture wasdiluted with DCM and washed with saturated aqueous NaHCO₃. The organicswere dried over Na₂SO₄, and the solvent removed. Half of material wasthen purified by preparative HPLC to yield the title compound (22 mg) asa TFA salt. MS m/z [M+H]⁺ calc'd for C₂₅H₂₅ClFN₃O₅, 502.15. found 502.1.

Example 4B:(2R,4R)-4-[(5-Acetyl-1H-pyrazole-3-carbonyl)amino]-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxypentanoicAcid

(2R,4R)-4-[(5-Acetyl-1H-pyrazole-3-carbonyl)amino]-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxypentanoicacid ethyl ester (75 mg, 150 μmol) was stirred at room temperature withLiOH (10.8 mg, 450 μmol) in water (450 μL, 25 mmol) and EtOH (450 μL,7.7 mmol), overnight. The solvent was removed and the remaining materialwas purified by preparative HPLC to yield the title compound (11.6 mg)as a TFA salt.

Example 5A:(2R,4R)-4-[(5-Acetyl-1H-pyrazole-3-carbonyl)amino]-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicAcid Ethyl Ester

To a stirred solution of 3-acetyl-1H-pyrazole-5-carboxylic acid (50.9mg, 330 μmol), HATU (125 mg, 330 μmol), and DIPEA (104 μL, 0.6 mmol) inDMF (0.6 mL, 7.7 mmol), was added(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (104 mg, 300 μmol). The resulting mixture was stirred at roomtemperature until the reaction was complete. The mixture was dilutedwith DCM and washed with saturated aqueous NaHCO₃. The organics weredried over Na₂SO₄, and the solvent removed. Half of material was thenpurified by preparative HPLC to yield the title compound (20 mg) as aTFA salt. MS m/z [M+H]⁺ calc'd for C₂₅H₂₆ClN₃O₅, 484.16. found 484.1.

Example 5B:(2R,4R)-4-[(5-Acetyl-1H-pyrazole-3-carbonyl)amino]-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicAcid

(2R,4R)-4-[(5-Acetyl-1H-pyrazole-3-carbonyl)amino]-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-pentanoicacid ethyl ester (72 mg, 150 μmol) was stirred at room temperature withLiOH (10.8 mg, 450 μmol) in water (450 μL, 25 mmol) and EtOH (450 μL,7.7 mmol), overnight. The solvent was removed and the remaining materialwas purified by preparative HPLC to yield the title compound (11.5 mg)as a TFA salt.

Example 6A:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-{[3-(2-fluorophenyl)isoxazole-5-carbonyl]amino}-2-hydroxpentanoicAcid

To a mixture of 3-(2-fluorophenyl)isoxazole-5-carboxylic acid (14.3 mg,69 μmol) and HATU (26.2 mg, 69 μmol) in DMF (0.5 mL, 6 mmol) at roomtemperature, was added(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (20.0 mg, 58 μmol) and DIPEA (20.0 μL, 115 μmol). The mixture wasstirred at room temperature overnight. MeOH (2.0 mL, 49 mmol), water(1.0 mL, 56 mmol), and LiOH monohydrate (9.7 mg, 230 μmol) was added,and the resulting mixture was stirred at room temperature for 1 hour.The resulting solids were filtered and rinsed with MeCN and water toyield the title compound (17.1 mg). MS m/z [M+H]⁺ calc'd forC₂₇H₂₂ClFN₂O₅, 509.12. found 509.

Example 6B:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-{[3-(2-fluorophenyl)isoxazole-5-carbonyl]amino}-2-hydroxypentanoicAcid Ethoxycarbonyloxymethyl Ester

A solution of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (HCl salt; 1.5 g, 4 mmol),3-(2-fluorophenyl)isoxazole-5-carboxylic acid (814 mg, 4 mmol), HOBt(1.1 g, 8 mmol), EDC (1.5 g, 8 mmol) and DIPEA (1.6 g, 12 mmol) in DMF(50 ml) was stirred for 15 hours at room temperature. The reaction wasquenched with water (50 mL) and extracted with EtOAc (3×60 mL), andorganic layers were collected and concentrated. The residue was purifiedby column chromatography (petroleum ether/EtOAc=5:1) to yield Compound 1(1.1 g). LC-MS: 537 [M+H]⁺.

A suspension of Compound 1 (200 mg, 0.37 mmol) and LiOH monohydrate (78mg, 1.9 mmol) in THF/MeOH/H₂O (2:2:1 20 mL) was stirred for 3 hours atroom temperature. The mixture was concentrated to remove the organicsolvents, and the residue was adjusted to pH 6 with aq. HCl (1 M). Themixture was filtered to yield Compound 2 as white solid (120 mg). LC-MS:509 [M+H]⁺.

A suspension Compound 2 (150 mg, 0.3 mmol), chloromethyl ethyl carbonate(84 mg, 0.6 mmol), NaI (15 mg, 0.6 mmol), and pyridine (96 mg, 1.2 mmol)in DMF (20 mL) was stirred for 15 hours at 30° C. The reaction wasquenched with water (15 mL) and extracted with EtOAc (3×30 mL). Thecombined organic layers were concentrated in vacuo and purified bycolumn chromatography (petroleum ether/EtOAc=3:1) to yield the titlecompound as an off-white solid (10 mg). LC-MS: 611 [M+H]⁺. ¹H NMR (400MHz, CDCl₃): δ 1.33 (t, J=7.2 Hz, 3H), 2.03-2.08 (m, 1H), 2.30-2.34 (m,1H), 3.05 (d, J=6.4 Hz, 2H), 4.26 (q, J=7.2 Hz, 2H), 4.48 (br, 1H), 4.75(br, 1H), 5.68-5.70 (m, 1H), 5.81-5.83 (m, 1H), 6.88 (d, J=8.8 Hz, 1H),7.28-7.34 (m, 7H), 7.42-7.53 (m, 4H), 7.96-7.99 (t, J=6.8 Hz, 1H).

Example 6C:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-{[3-(2-fluorophenyl)isoxazole-5-carbonyl]amino}-2-hydroxypentanoicAcid Isopropoxycarbonyloxymethyl Ester

To a stirred solution of(2R,4R)-4-(t-butoxycarbonylamino)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicacid (2.4 g, 5.7 mmol) in DMF (50 mL) was added chloromethyl isopropylcarbonate (1.3 g, 8.6 mmol). To this mixture, NaI (1.71 g, 11.4 mmol)and DIPEA (2.2 g, 17.1 mmol) were added and the resulting mixture wasstirred at room temperature overnight. Water (100 mL) was added and themixture was extracted with EtOAc (3×80 mL). The combined organic layerswere washed with saturated aqueous NaCl (2×100 mL) and water (2×100 mL),dried over anhydrous Na₂SO₄, and concentrated in vacuo. The product waspurified by column chromatography (petroleum ether/EtOAc, 15:1-6:1) toyield Compound 1 as a white solid (574 mg). LC-MS: 558 [M+Na]⁺.

Compound 1 (574 mg, 1.1 mmol) was dissolved in an HCl-dioxane solution(1.4 M, 50 mL) at 0° C., and the resulting mixture was stirred at roomtemperature for 8 hours. After removal of the solvent, the residue wasdispersed in EtOAc (10 mL). The precipitate was collected by filtrationto yield Compound 2 as a white solid HCl salt (300 mg). LC-MS: 436[M+H]⁺.

To a solution of Compound 2 (150 mg, 340 μmol) in DMF (10 mL) was added3-(2-fluorophenyl)-isoxazole-5-carbonyl chloride (79 mg, 380 μmol) andDIPEA (120 μL, 680 μmol). The mixture was stirred at room temperatureovernight. Water (50 mL) was added, and the mixture was then extractedwith EtOAc (3×30 mL). The combined organic layers were washed withsaturated aqueous NaCl (2×50 mL) and water (2×50 mL), dried overanhydrous Na₂SO₄, and concentrated in vacuo. The residue was thenpurified by preparative HPLC (CAN-H₂O (0.1% TFA), 75-80) to yield thetitle compound as a white solid (60 mg). LC-MS: 625 [M+H]⁺. ¹H-NMR (400MHz, DMSO-d₆): δ 1.22-1.23 (m, 6H), 2.01-2.07 (m, 2H), 2.89-2.96 (m,2H), 4.37-4.38 (m, 2H), 4.80 (br, 1H), 5.67-5.69 (m, 2H), 5.71-5.78 (m,1H), 7.31-7.33 (m, 7H), 7.42-7.43 (m, 5H), 7.61-7.63 (m, 1H), 8.95 (d,J=6.8 Hz, 1H).

Example 6D:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-{[3-(2-fluorophenyl)isoxazole-5-carbonyl]amino}-2-hydroxypentanoicAcid Acetoxymethyl Ester

To a stirred solution of bromomethyl acetate (977 mg, 6.4 mmol) in DMF(10 mL) was added dropwise a solution of(2R,4R)-4-(t-butoxycarbonylamino)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicacid (1.8 g, 4.3 mmol) in DMF (10 mL) at room temperature. Pyridine (1.5g, 19.2 mmol) was then added dropwise and the resulting mixture wasstirred at room temperature overnight. Water (150 mL) was added and themixture was extracted with EtOAc (3×100 mL). The combined organic layerswere washed with saturated aqueous NaCl (2×100 mL) and water (3×100 mL),dried over anhydrous Na₂SO₄, and concentrated in vacuo. The crudeproduct was purified by column chromatography (petroleumether/EtOAc=6:1) to yield Compound 1 as a white solid (570 mg. LC-MS:436 [M-tBu+H]⁺.

Compound 1 (570 mg, 1.2 mmol) was dissolved in an HCl-dioxane solution(1.4 mol/L, 50 mL) at 0° C. The resulting mixture was stirred at roomtemperature for 8 hours, then concentrated in vacuo. The residue wasdispersed in EtOAc (10 mL). The precipitate was collected by filtrationto yield Compound 2 as a white solid HCl salt (200 mg). LC-MS: 392[M+H]⁺.

To a solution of Compound 2 (150 mg, 380 μmol) in THF (10 mL) was added3-(2-fluorophenyl)-isoxazole-5-carbonyl chloride (79 mg, 380 μmol) andDIPEA (120 μL, 680 μmol). The mixture was stirred at room temperatureovernight. Water (50 mL) was added, and the mixture was then extractedwith EtOAc (3×20 mL). The organic layers were washed with saturatedaqueous NaCl (2×30 mL) and water (2×30 mL), dried over anhydrous Na₂SO₄,and concentrated in vacuo. The residue was then purified by preparativeHPLC (CAN-H₂O (0.1% TFA), 75-80) to yield the title compound as a whitesolid (13 mg). LC-MS: 581 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 1.90-1.99(m, 2H), 2.07 (s, 3H), 2.89-2.96 (m, 2H), 4.27-4.38 (m, 2H), 5.67-5.69(m, 2H), 5.78 (br, 1H), 7.31-7.33 (m, 7H), 7.42-7.43 (m, 5H), 7.61-7.63(m, 1H), 8.95 (d, J=8.8 Hz, 1H).

Example 6E:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-{[3-(2-fluorophenyl)isoxazole-5-carbonyl]amino}-2-hydroxpentanoicAcid (S)-2-Methoxycarbonylamino-3-methylbutyryloxymethyl Ester

2,6-Lutidine (96 mg, 0.9 mmol) was added to a solution of(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-4-{[3-(2-fluorophenyl)isoxazole-5-carbonyl]amino}-2-hydroxypentanoicacid (150 mg, 0.3 mmol), (S)-2-methoxycarbonylamino-3-methylbutyric acidchloromethyl ester (134 mg, 0.6 mmol) and NaI (135 mg, 0.9 mmol) in DMF(10 mL). The mixture was stirred at 50° C. overnight. Water (20 mL) wasadded, and the mixture was extracted with ethyl ether (2×30 mL). Thecombined organic layers were dried over anhydrous Na₂SO₄ andconcentrated in vacuo. The residue was purified by column chromatography(petroleum ether/EtOAc=4/1˜1/2) to yield the title compound as anoff-yellow solid (8 mg). LC-MS: 695.9 [M+H]⁺. ¹H-NMR (CD₃OD-d₄, 400 MHz)δ 0.92-0.95 (m, 6H), 2.03-2.08 (m, 3H), 3.00-3.10 (m, 2H), 4.09 (br,1H), 4.27-4.28 (m, 1H), 4.87 (br, 1H), 5.75-5.85 (m, 2H), 7.26-7.37 (m,6H), 7.51-7.53 (m, 6H), 7.93-7.95 (m, 1H).

Example 7A:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-methoxyisoxazole-5-carbonyl)amino]pentanoicAcid

3-Methoxyisoxazole-5-carboxylic acid (39.4 mg, 275 μmol) and HCTU (128mg, 310 μmol) were combined in DMF, and stirred for 10 minutes at roomtemperature. DIPEA (72 μL, 413 μmol) and(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (50 mg, 0.1 mmol) were added, and the resulting mixture wasstirred at room temperature overnight. The mixture was combined withEtOH (402 jL, 6.9 mmol) and 1 M LiOH in water (1.1 mL, 1.1 mmol). Themixture was stirred at room temperature for 1 hour, evaporated underreduced pressure, and purified by preparative HPLC to yield the titlecompound. MS m/z [M+H]⁺ calc'd for C₂₂H₂₁ClN₂O₆, 445.11. found 445.2.

Example 7B:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-methoxyisoxazole-5-carbonyl)amino]pentanoicAcid 2-Methoxycarbonylamino-3-methylbutyryloxymethyl Ester

To a solution of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (HCl salt, 1 g, 2.6 mmol) in THF (30 mL) was added a solution of3-methoxyisoxazole-5-carbonyl chloride (430 mg, 3 mmol) in THF (10 mL)and Et₃N (790 mg, 7.8 mmol) at 0° C. The resulting solution was stirredat room temperature for 6 hours, then concentrated in vacuo to yield ayellow solid which was purified by chromatography (PE:EtOAc=8:1) toyield Compound 1 as a white solid (0.9 g). LC-MS: 473 [M+H]⁺.

To a solution of Compound 1 (0.5 g, 1 mmol) in MeOH/THF/H₂O (15 mL/15mL/3 mL) was added LiOH (120 mg, 5 mmol). The resulting mixture wasstirred at room temperature for 5 hours, then concentrated. The pH ofthe mixture was adjusted to pH 5 with HCl (1N, 20 mL). The solids werefiltered to yield Compound 2 as a white solid (0.4 g). LC-MS: 445[M+H]⁺.

DIPEA (250 mg, 2 mmol) was added to a solution of Compound 2 (220 mg,0.5 mmol), (S)-2-methoxycarbonylamino-3-methylbutyric acid chloromethylester (220 mg, 1 mmol) and NaI (300 mg, 2 mmol) in DMF (5 mL). Themixture was stirred at room temperature for 12 hours. The mixture wasthen poured into water (20 mL), extracted with EtOAc (3×15 mL), washedwith saturated aqueous NaCl (30 mL), dried over anhydrous Na₂SO₄, andconcentrated in vacuo. The residue was then purified by chromatography(petroleum ether:EtOAc=6:1) to yield the title compound as a white solid(10 mg). LC-MS: 632 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 0.86 (d, J=6.8Hz, 6H), 1.97-2.02 (m, 3H), 2.92-2.94 (m, 2H), 3.92 (s, 3H), 4.36-4.21(m, 2H), 5.70-5.79 (m, 2H), 6.72 (s, 1H), 7.29 (d, J=8.0 Hz, 2H),7.46-7.61 (m, 2H), 7.60-7.69 (m, 4H), 8.73 (d, J=8.8 Hz, 1H).

Example 7C:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-methoxyisoxazole-5-carbonyl)amino]pentanoicAcid Isopropoxycarbonyloxymethyl Ester

A mixture of(2R,4R)-4-(t-butoxycarbonylamino)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicacid (600 mg, 1.4 mmol), chloromethyl isopropyl carbonate (426 mg, 2.8mmol), NaI (420 mg, 2.8 mmol) and DIPEA (546 mg, 4.2 mmol) in DMF (30.0mL) was stirred at 25° C. overnight. The reaction mixture was pouredinto water (60 mL) and extracted with EtOAc (3×50 mL). The combinedorganic layers were washed with saturated aqueous NaCl (2×50 mL) andwater (2×50 mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo.The product was purified by column chromatography (petroleumether:EtOAc=2:1) to yield Compound 1 as a colorless liquid (240 mg).LC-MS: 480 [M-tBu+H]+.

Compound 1 (240 mg, 0.45 mol) was dissolved in an HCl-dioxane solution(10.0 mL, 3.0 M) at 0° C., and the resulting mixture was stirred at roomtemperature overnight. The solvent was removed in vacuo. The residue wasdispersed in EtOAc (10 mL). The precipitate was collected by filtrationto yield Compound 2 as a white solid HCl salt (80 mg). LC-MS: 436[M+H]⁺.

A mixture of Compound 2 (70 mg, 0.15 mmol),3-methoxyisoxazole-5-carboxylic acid (43 mg, 0.3 mmol), HOBT (40 mg, 0.3mmol), EDCI (57 mg, 0.3 mmol) and DIPEA (78 mg, 0.6 mmol) in DMF (10.0mL) was stirred at room temperature overnight. The mixture was pouredinto water (30 mL) and extracted with EtOAc (3×30 mL). The organic layerwas separated, washed with saturated aqueous NaCl (2×30 mL) and water(2×30 mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo. Theresidue was purified by column chromatography (petroleumether:EtOAc=2:1) to yield the title compound as a white solid (20 mg).LC-MS: 561 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 1.30 (d, J=6.4 Hz, 6H),2.04-2.05 (m, 1H), 2.25-2.29 (m, 1H), 2.99-3.01 (m, 2H), 3.48 (br, 1H),4.00 (s, 3H), 4.46-4.48 (m, 1H), 4.50 (br, 1H), 4.92-4.93 (m, 1H),5.69-5.79 (m, 2H), 6.51 (s, 1H), 6.79-6.70 (m, 1H), 7.28-7.57 (m, 8H).

Example 7D:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-methoxyisoxazole-5-carbonyl)amino]pentanoicAcid Acetoxymethyl Ester

Bromomethyl acetate (977 mg, 6.4 mmol) was added to a stirred solution(2R,4R)-4-(t-butoxycarbonylamino)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicacid (1.8 g, 4.3 mmol) in DMF (20 mL) at room temperature. Pyridine (1.7g, 21.5 mmol) was added dropwise and the resulting mixture was stirredat room temperature overnight. Water (50 mL) was added and the mixturewas extracted with EtOAc (3×50 mL). The combined organic layers werewashed with saturated aqueous NaCl (2×60 mL) and water (2×60 mL), driedover anhydrous Na₂SO₄, and concentrated in vacuo. The product waspurified by column chromatography (petroleum ether:EtOAc, 15:1-6:1) toyield Compound 1 as a white solid (570 mg). LC-MS: 436 [M-tBu+H]⁺.

Compound 1 (570 mg, 1.2 mmol) was added to an HCl-dioxane solution (1.4mol/L, 50 mL) at 0° C., and the resulting mixture was stirred at roomtemperature for 2 hours then concentrated in vacuo. The residue wasdispersed in EtOAc (10 mL). The precipitate was collected by filtrationto yield Compound 2 as a white solid HCl salt (200 mg). LC-MS: 392[M+H]⁺.

To a solution of Compound 2 (100 mg, 255 μmol) in DMF (10 mL) was added3-methoxyisoxazole-5-carboxylic acid (79 mg, 383 μmol), HOBt (69 mg, 510μmol), EDCI (98 mg, 510 μmol) and DIPEA (0.2 mL, 1.0 mmol). The mixturewas stirred at room temperature overnight. Water (30 mL) was added andthe mixture was extracted with EtOAc (3×30 mL). The combined organiclayers were washed with saturated aqueous NaCl (2×30 mL) and water (2×30mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo. The residuewas purified by preparative HPLC (CAN-H₂O (0.1% TFA), 60-65) to yieldthe title compound as a white solid (15 mg). LC-MS: 517 [M+H]⁺. ¹H-NMR(DMSO-d₆, 400 Hz): δ 1.84-1.99 (m, 2H), 2.07 (s, 3H), 2.89-2.96 (m, 2H),3.92 (s, 3H), 4.21-4.32 (m, 2H), 5.66-5.69 (m, 2H), 5.74 (br, 1H), 6.72(s, 1H), 7.29 (d, J=8.4 Hz, 2H), 7.28-7.30 (m, 2H), 7.60-7.62 (m, 3H),7.69 (s, 1H), 8.76 (d, J=8.8 Hz, 1H).

Example 7E:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-methoxyisoxazole-5-carbonyl)-amino]pentanoicAcid Ethoxycarbonyloxymethyl Ester

To a stirred solution of chloromethyl ethyl carbonate (0.2 mL, 1.1 mmol)in DMF (1 mL) was added dropwise a solution of(2R,4R)-4-(t-butoxycarbonylamino)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicacid (400 mg, 960 μmol) in DMF (6 mL) at room temperature. NaI (432 mg,2.88 mmol) and DIPEA (0.5 mL, 2.88 mmol) were added and the resultingmixture was stirred at room temperature overnight. Water (50 mL) wasadded and the mixture was extracted with EtOAc (3×30 mL). The combinedorganic layers were washed with saturated aqueous NaCl (2×50 mL) andwater (2×50 mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo.The product was purified by column chromatography (petroleumether:EtOAc, 15:1-6:1) to yield Compound 1 as a white solid (190 mg).LC-MS: 544 [M+Na]⁺.

Compound 1 (190 mg, 360 μmol) was added to an HCl-dioxane solution (1.4mol/L, 50 mL) at 0° C., and the resulting mixture was stirred at roomtemperature for 2 hours then concentrated in vacuo. The residue wasdispersed in EtOAc (10 mL). The precipitate was collected by filtrationto yield Compound 2 as a white solid HCl salt (150 mg). LC-MS: 422[M+H]⁺.

To a stirred solution of Compound 2 (150 mg, 360 μmol) in THF (20 mL)was added dropwise a solution of 3-methoxyisoxazole-5-carbonyl chloride(73 mg, 530 μmol) in THF (2 mL) at room temperature. DIPEA (0.2 mL) wasadded dropwise and the resulting mixture was stirred at room temperatureovernight. Water (50 mL) was added and the mixture was extracted withEt₂O (3×40 mL). The combined organic layers were washed with saturatedaqueous NaCl (2×50 mL) and water (2×50 mL), dried over anhydrous Na₂SO₄,and concentrated in vacuo. The residue was purified by preparative HPLC(CAN-H₂O (0.1% TFA), 60-70) to yield the title compound as a white solid(25 mg). LC-MS: 546.9 [M+H]⁺. ¹H-NMR (DMSO-d₆, 400 MHz): δ 1.22 (t,J=6.8 Hz, 3H), 1.87-1.96 (m, 2H), 2.83-2.89 (m, 2H), 3.92 (s, 3H), 4.16(q, J=6.8 Hz, 2H), 4.18-4.20 (m, 2H), 5.68-5.78 (m, 3H), 6.72 (s, 1H),7.29 (d, J=8.0 Hz, 2H), 7.45-7.48 (m, 2H), 7.60-7.62 (m, 3H), 7.69 (m,1H), 8.76 (d, J=8.8 Hz, 1H).

Example 8A:(2R,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-[(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

(2R,4R)-5-(4-Bromophenyl)-2-hydroxy-4-[(1-hydroxy-1H-1,2,3-triazole-4-carbonyl)amino]pentanoicacid ethyl ester (30 mg, 70 μmol) was combined with5-chloro-2-fluorophenylboronic acid (22 mg, 126 μmol), K₂CO₃ (29.1 mg,211 μmol), EtOH (0.8 mL, 10 mmol), and water (0.2 mL, 10 mmol).SilicaCat DPP-Pd (0.28 mmol/g loading; 25.1 mg, 7.0 μmol) was added thelast. The resulting mixture was microwaved at 100° C. for 10 minutes. 1M LiOH in water (281 μL, 281 μmol) was added, and the resulting mixturewas stirred for 1 hour. The mixture was filtered, concentrated, andpurified by preparative HPLC to yield the title compound (17 mg). MS m/z[M+H]⁺ calc'd for C₂₀H₁₈ClFN₄O₅, 449.10. found 449.2.

Example 8B:(2R,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-[(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid 2,2,3,3,3-pentafluoropropyl Ester

(2R,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxypentanoicacid 2,2,3,3,3-pentafluoropropyl ester (32.1 mg, 68.3 μmol),1-hydroxy-1H-[1,2,3]triazole-4-carboxylic acid (10.6 mg, 82 μmol) andHATU (39.0 mg, 102 μmol) were stirred in DMF (1.0 mL, 12.9 mmol) for 10minutes. DIPEA (35.7 μL, 205 μmol) was added dropwise and the resultingmixture was stirred at room temperature for 1 hour. The mixture wasconcentrated and to yield a clear yellow liquid, which was purifiedusing preparative scale C18 column chromatography (small column; 30-90%MeCN in water with 0.05% TFA) to yield the title compound as a whitesolid (4.9 mg). MS m/z [M+H]⁺ calc'd for C₂₃H₁₉ClF₆N₄O₅, 581.09. found581.

Example 8C:(2R,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-[(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid 5-Methyl-2-oxo-[1,3]dioxol-4-ylmethyl Ester

(2R,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxypentanoicacid 5-methyl-2-oxo-[1,3]dioxol-4-ylmethyl ester (30.7 mg, 68.3 μmol),1-hydroxy-1H-[1,2,3]triazole-4-carboxylic acid (10.6 mg, 82 μmol) andHATU (39.0 mg, 102 μmol) were stirred in DMF (1.0 mL, 12.9 mmol) for 10minutes. DIPEA (35.7 μL, 205 μmol) was added dropwise and the resultingmixture was stirred at room temperature for 1 hour. The mixture wasconcentrated and to yield a clear yellow liquids, which was purifiedusing preparative scale C18 column chromatography (small column; 30-90%MeCN in water with 0.05% TFA) to yield the title compound as a whitesolid (27.1 mg). MS m/z [M+H]⁺ calc'd for C₂₅H₂₂ClFN₄O₈, 561.11. found561.1.

Example 8D:(2R,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-{[1-(5-methyl-2-oxo-[1,3]dioxol-4-ylmethoxy)-1H-[1,2,3]triazole-4-carbonyl]amino}pentanoicAcid

4-Chloromethyl-5-methyl-1,3-dioxol-2-one (11.0 μL, 100 μmol) was addedto a solution of(2R,4R)-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-[(1-hydroxy-1H-1,2,3-triazole-4-carbonyl)amino]pentanoicacid (30.0 mg, 66.8 μmol) in acetone (0.5 mL, 6.8 mmol). Et₃N (18.6 μL,134 μmol) was added and the resulting mixture was stirred at 65° C. for2 hours. The mixture was concentrated in vacuo to yield a yellow solid.The crude solid was purified by preparative scale HPLC (C18 columnchromatography, small column, using 30-90% MeCN in water with 0.05% TFA)to yield the title compound as a white solid (6.5 mg). MS m/z [M+H]⁺calc'd for C₂₅H₂₂ClFN₄O₅, 561.11. found 561.1.

Example 8E:(2R,4R)-4-[(1-Butyryloxymethoxy-1H-[1,2,3]triazole-4-carbonyl)amino]-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxypentanoicAcid

Chloromethyl butyrate (13.7 mg, 100 μmol) was added to a solution of(2R,4R)-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-[(1-hydroxy-1H-1,2,3-triazole-4-carbonyl)amino]pentanoicacid (30.0 mg, 66.8 μmol) in acetone (0.5 mL, 6.8 mmol). Et₃N (18.6 μL,134 μmol) was added and the resulting mixture was stirred at 65° C. for2 hours. The mixture was then concentrated in vacuo to yield a yellowliquid. The crude liquid was purified (preparative scale C18 columnchromatography, small column, using 30-90% MeCN in water with 0.05% TFA)to yield the title compound as a white solid (6.0 mg). MS m/z [M+H]⁺calc'd for C₂₅H₂₆ClFN₄O₇, 549.15. found 549.1.

Example 8F:(2R,4R)-4-{[1-((S)-2-Amino-3-methylbutyryloxymethoxy)-1H-[1,2,3]triazole-4-carbonyl]-amino}-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxypentanoicAcid

(S)-2-t-Butoxycarbonylamino-3-methylbutyric acid chloromethyl ester(44.4 mg, 167 μmol) was added to a solution of(2R,4R)-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-[(1-hydroxy-1H-1,2,3-triazole-4-carbonyl)amino]pentanoicacid (30.0 mg, 66.8 μmol) in acetone (0.5 mL, 6.8 mmol). Et₃N (18.6 μL,134 μmol) was added and the resulting mixture was stirred at 65° C. for2 hours. 1:1 TFA/DCM (1.0 mL, 6.2 mmol) was added and the mixture wasstirred at room temperature for 30 minutes, and then concentrated invacuo to yield a yellow liquid. The crude liquid was purified(preparative scale C18 column chromatography, small column, using 30-90%MeCN in water with 0.05% TFA) to yield the title compound as a whitesolid (10 mg). MS m/z [M+H]⁺ calc'd for C₂₆H₂₉ClFN₅O₇, 578.17. found578.1.

Example 8G:(2R,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-{[1-((S)-2-methoxycarbonylamino-3-methylbutyryloxymethoxy)-1H-[1,2,3]triazole-4-carbonyl]amino}pentanoicAcid

(S)-2-Methoxycarbonylamino-3-methylbutyric acid chloromethyl ester (37.4mg, 167 μmol) was added to a solution of(2R,4R)-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-[(1-hydroxy-1H-1,2,3-triazole-4-carbonyl)amino]pentanoicacid (30.0 mg, 66.8 μmol) in acetone (0.5 mL, 6.8 mmol). Et₃N (18.6 μL,134 μmol) was added and the resulting mixture was stirred at 65° C. for2 hours. The mixture was then concentrated in vacuo to yield a yellowliquid. The crude liquid was purified (preparative scale C18 columnchromatography, small column, using 30-90% MeCN in water with 0.05% TFA)to yield the title compound as a white solid (8.2 mg). MS m/z [M+H]⁺calc'd for C₂₈H₃₁ClFN₅O₉, 636.18. found 636.1.

Example 8H:(2R,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-[(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid Butyryloxymethyl Ester

DIPEA (35.8 μL, 206 μmol) was added to a solution of(2R,4R)-4-amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxypentanoicacid butyryloxymethyl ester (30.0 mg, 68.5 μmol),1-hydroxy-1H-1,2,3-triazole-4-carboxylic acid (13.3 mg, 103 μmol) andHATU (39.1 mg, 103 μmol) in DMF (500 μL, 6.5 mmol). The resultingmixture was stirred at room temperature for 30 minutes, thenconcentrated in vacuo to yield a clear yellow liquid. The crude liquidwas purified by preparative scale HPLC (C18 column chromatography, smallcolumn, using 30-90% MeCN in water with 0.05% TFA) to yield the titlecompound as a white solid (9.0 mg). MS m/z [M+H]⁺ calc'd forC₂₅H₂₆ClFN₄O₇, 549.15. found 549.1.

Example 8I:(2R,4R)-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-[(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid Ethoxycarbonyloxymethyl Ester

To a solution of 1-allyloxy-1H-[1,2,3]triazole-4-carboxylic acid ethylester (1.7 g, 8.6 mmol) in MeOH (20 mL) was added a solution of LiOH(1.1 g, 45.3 mmol) in water (4 mL). The mixture was stirred at roomtemperature until the reaction was complete (1 hour). The mixture wasconcentrated, diluted with water (10 mL), and extracted with EtOAc (2×20mL). The aqueous layer was acidified by 1N HCl to pH=3, extracted withEtOAc (3×30 mL), and the combined organic layers were washed withsaturated aqueous NaCl (30 mL) and dried over anhydrous Na₂SO₄. Thesolution was evaporated to yield Compound 1 (1.3 g) as a white solid.LC-MS: 170 [M+H]⁺.

To a solution of(2R,4R)-4-amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxypentanoicacid ethyl ester (HCl salt) (800 mg, 2.0 mmol) and Compound 1 (337 mg,2.0 mmol) in DMF (15 mL) were added PyBOP (1.0 g, 2.0 mmol) and DIPEA(987 μL, 6.0 mmol) at 0° C. The mixture was stirred at room temperaturefor 4 hours. Water (50 mL) was added, and the mixture was extracted withEtOAc (3×10 mL), and the combined organic layers were washed withsaturated aqueous NaCl and dried over anhydrous Na₂SO₄. The solution wasconcentrated to give a crude product which was purified by silica gelchromatography (silica gel: 200-300 mesh; eluted with PE:EtOAc=10:1 to5:1 to 1:1) to yield Compound 2 (700 mg) as a light yellow solid. LC-MS:517 [M+H]⁺.

To a solution of Compound 2 (700 mg, 1.4 mmol) in MeOH (15 mL) and water(3 mL) was added LiOH (171 mg, 4.1 mmol) at room temperature. Themixture was stirred at room temperature overnight. The mixture wasconcentrated, water (20 mL) was added, and the mixture was extractedwith EtOAc (2×10 mL). The aqueous layer was acidified by 1N HCl to pH=3,extracted with EtOAc (3×10 mL), and the combined organic layers werewashed with saturated aqueous NaCl (30 mL) and dried over anhydrousNa₂SO₄. The solution was evaporated to yield Compound 3 (650 mg) as ayellow solid. LC-MS: 489 [M+H]⁺.

To a mixture of Compound 3 (80 mg, 160 μmol) and chloromethyl ethylcarbonate (1.0 ml) were added NaI (48 mg, 0.32 mmol) and lutidine (52mg, 480 μmol). The mixture was stirred at 50° C. for 6 hours. Themixture was then cooled to room temperature, diluted with water (15 ml),and extracted with EtOAc (2×15 mL). The combined organic layers werewashed with saturated aqueous NaCl (20 mL), dried over anhydrous Na₂SO₄,concentrated, and purified by silica gel chromatography (silica gel:200-300 mesh; elute with PE:EtOAc from 4:1 to 1:1) to yield Compound 4(60 mg) as a light yellow solid. LC-MS: 591 [M+H]⁺.

To a solution of Compound 4 (40 mg, 68 μmol) in MeCN (10 mL) was addedNaI (10 mg, 68 μmol), and the mixture was stirred at room temperaturefor 5 minutes. To this stirred suspension, TMSCl (11 mg, 102 μmol) wasadded and stirred for another 10 minutes. The reaction was quenched bysodium thiosulphate and the mixture was extracted with EtOAc (2×10 mL).The combined organic layers were washed with saturated aqueous NaCl (20mL) and dried over anhydrous Na₂SO₄. The residue was purified bypre-HPLC [Daisogel-C18, 250×50 mm, 10 μm; CAN-H₂O (0.1% TFA) from 60% to90%] to yield the title compound (8 mg) as a white solid. LC-MS: 551[M+H]⁺. ¹H-NMR (CDCl₃, 400 Hz): δ 1.27 (t, 3H), 2.06-2.11 (m, 1H),2.14-2.24 (m, 1H), 2.96-3.01 (m, 2H), 4.18 (q, 2H), 4.42 (m, 1H), 4.71(m, 1H), 5.57-5.65 (m, 2H), 7.05 (t, 1H), 7.22-7.24 (m, 2H), 7.35-7.40(m, 4H), 8.04 (s, 1H).

Example 8J:(2R,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-[(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid Isopropoxycarbonyloxymethyl Ester

The title compound was prepared (110 mg) using the procedures describedherein.

Example 8K:(2R,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-[(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid 1-Cyclohexyloxycarbonyloxyethyl Ester

The title compound was prepared (16.6 mg) using the procedures describedherein.

Example 8L:(2R,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-[(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicacid (S)-2-methoxycarbonylamino-3-methyl-butyryloxymethyl Ester

The title compound was prepared (117 mg) using the procedures describedherein.

Example 8M:(2R,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-[(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid Acetoxymethyl Ester

Using the procedures described herein, the title compound can also beprepared.

Example 9A:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-oxo-1-phenyl-4,5-dihydro-1H-[1,2,4]triazole-3-carbonyl)amino]pentanoicAcid

5-Oxo-1-phenyl-4,5-dihydro-1H-[1,2,4]triazole-3-carboxylic acid (42.3mg, 206 μmol) and HCTU (128 mg, 310 μmol) were combined in DMF, andstirred for 10 minutes at room temperature. DIPEA (72 μL, 413 μmol) and(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (50 mg, 0.1 mmol) were added, and the resulting mixture wasstirred at room temperature overnight. The mixture was combined withEtOH (402 μL, 6.9 mmol) and 1 M LiOH in water (1.1 mL, 1.1 mmol). Themixture was stirred at room temperature for 1 hour, evaporated underreduced pressure, and purified by preparative HPLC to yield the titlecompound (1.8 mg) as a TFA salt. MS m/z [M+H]⁺ calc'd for C₂₆H₂₃ClN₄O₅,507.14. found 507.2.

Example 9B:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-oxo-1-phenyl-4,5-dihydro-1H-[1,2,4]triazole-3-carbonyl)amino]pentanoicAcid Ethoxycarbonyloxymethyl Ester

A solution of(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-oxo-1-phenyl-4,5-dihydro-1H-[1,2,4]triazole-3-carbonyl)amino]pentanoicacid (253 mg, 0.5 mmol), chloromethyl ethyl carbonate (69 mg, 0.5 mmol),2,6-lutidine (165 mg, 1.5 mmol) and NaI (150 mg, 1 mmol) in DMF (20 mL)was stirred for 15 hours at 50° C. The mixture was quenched with water(20 mL) and extracted with EtOAc (3×30 mL). The combined organic layerswere collected and concentrated in vacuo. The residue was purified bycolumn chromatography (petroleum ether/EtOAc=3:1) to yield the titlecompound as a white solid (13 mg). LC-MS: 608.8 [M+H]⁺. ¹H NMR: (CDCl₃,400 MHz) δ 1.27 (t, J=8.0 Hz, 3H), 2.03-2.33 (m, 2H), 3.02-3.05 (m, 2H),4.18 (q, J=8.0 Hz, 2H), 4.49 (br, 1H), 4.85 (br, 1H), 5.57-5.76 (m, 2H,7.03 (d, J=12 Hz, 1H), 7.25-7.40 (m, 4H), 7.42-7.51 (m, 3H), 7.53-7.61(m, 3H), 7.93 (d, J=8.0 Hz, 2H).

Example 9C:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-oxo-1-phenyl-4,5-dihydro-1H-[1,2,4]triazole-3-carbonyl)amino]pentanoicacid (S)-2-methoxycarbonylamino-3-methylbutyryloxymethyl Ester

Using the procedures described herein, the title compound can also beprepared.

Example 10A:(2R,4R)-4-[(5-Acetyl-2-phosphonooxymethyl-2H-pyrazole-3-carbonyl)amino]-5-biphenyl-4-yl-2-hydroxypentanoicAcid Ethyl Ester

(2R,4R)-4-Amino-5-biphenyl-4-yl-2-hydroxypentanoic acid ethyl ester (HClsalt; 500 mg, 1 mmol, 1.0 eq.), 5-acetyl-2H-pyrazole-3-carboxylic acid(330.4 mg, 2.1 mmol, 1.5 eq.), and HATU (820 mg, 2.1 mmol, 1.5 eq.) werecombined in DMF (5 mL), and the resulting mixture was stirred for 2minutes. DIPEA (750 μL) was added and the mixture was stirred for 1hour. The mixture was dried under vacuum, and the product was purifiedusing reverse phase chromatography (10-70% MeCN/H₂O; 0.05% TFA over 70minutes) to yield Compound 1 as a TFA salt (300 mg, purity 98%). MS m/z[M+H]⁺ calc'd for C₂₅H₂₇N₃O₅, 450.20. found 450.2.

To a solution of Compound 1 (15.0 mg, 33.4 μmol) in DMF (103 μL, 1.3mmol) was added K₂CO₃ (5.1 mg, 36.7 μmol) and phosphoric acid di-t-butylester chloromethyl ester (9.50 mg, 36.7 μmol). The resulting mixture wasstirred at room temperature overnight, then evaporated under reducedpressure. The residue was diluted in EtOAc and a solution of 1M HCl wasadded to bring the pH to 4-5. The organic layer was extracted twice withEtOAc, washed with water followed by saturated aqueous NaCl, dried overanhydrous MgSO₄, filtered, and evaporated. The residue was purified byflash chromatography (30-90% EtOAc in hexanes). DCM (64.2 μL, 1.0 mmol)was added, followed by TFA (40 μL, 0.6 mmol), and the resulting mixturewas stirred at room temperature for 20 minutes. The mixture wasevaporated and purified by purified by preparative HPLC to yield thetitle compound as a TFA salt (4 mg). MS m/z [M+H]⁺ calc'd forC₂₆H₃₀N₃O₉P, 560.17. found 560.1.

Example 10B:(2R,4R)-4-[(5-Acetyl-2H-pyrazole-3-carbonyl)amino]-2-((S)-2-amino-3-methylbutyryloxy)-5-biphenyl-4-yl-pentanoicAcid Ethyl Ester

Using the procedures described herein, the title compound can also beprepared.

Example 10C:(2R,4R)-4-{[5-Acetyl-2-((S)-2-amino-3-methylbutyryloxymethyl)-2H-pyrazole-3-carbonyl]amino}-5-biphenyl-4-yl-2-hydroxypentanoicAcid Ethyl Ester

Using the procedures described herein, the title compound can also beprepared.

Example 11A:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(1H-tetrazole-5-carbonyl)amino]pentanoicAcid Ethoxycarbonyloxymethyl Ester

Using the procedures described herein, the title compound can also beprepared.

Assay In Vitro Assays for the Quantitation of Inhibitor Potencies (IC₅₀)at Human and Rat NEP, and Human ACE

The inhibitory activities of compounds at human and rat neprilysin (EC3.4.24.11; NEP) and human angiotensin converting enzyme (ACE) weredetermined using in vitro assays as described below.

Extraction of NEP Activity from Rat Kidneys

Rat NEP was prepared from the kidneys of adult Sprague Dawley rats.Whole kidneys were washed in cold phosphate buffered saline (PBS) andbrought up in ice-cold lysis buffer (1% Triton X-114, 150 mM NaCl, 50 mMtris(hydroxymethyl)aminomethane (Tris) pH 7.5; Bordier (1981) J. Biol.Chem. 256: 1604-1607) in a ratio of 5 mL of buffer for every gram ofkidney. Samples were homogenized on ice using a polytron hand heldtissue grinder. Homogenates were centrifuged at 1000×g in a swingingbucket rotor for 5 minutes at 3° C. The pellet was resuspended in 20 mLof ice cold lysis buffer and incubated on ice for 30 minutes. Samples(15-20 mL) were then layered onto 25 mL of ice-cold cushion buffer (6%w/v sucrose, 50 mM pH 7.5 Tris, 150 mM NaCl, 0.06%, Triton X-114),heated to 37° C. for 3-5 minutes and centrifuged at 1000×g in a swingingbucket rotor at room temperature for 3 minutes. The two upper layerswere aspirated off, leaving a viscous oily precipitate containing theenriched membrane fraction. Glycerol was added to a concentration of 50%and samples were stored at −20° C. Protein concentrations werequantitated using a BCA detection system with bovine serum albumin (BSA)as a standard.

Enzyme Inhibition Assays

Recombinant human NEP and recombinant human ACE were obtainedcommercially (R&D Systems, Minneapolis, Minn., catalog numbers 1182-ZNand 929-ZN, respectively). The fluorogenic peptide substrateMca-D-Arg-Arg-Leu-Dap-(Dnp)-OH (Medeiros et al. (1997) Braz. J. Med.Biol. Res. 30:1157-62; Anaspec, San Jose, Calif.) andAbz-Phe-Arg-Lys(Dnp)-Pro-OH (Araujo et al. (2000) Biochemistry39:8519-8525; Bachem, Torrance, Calif.) were used in the NEP and ACEassays respectively.

The assays were performed in 384-well white opaque plates at 37° C.using the fluorogenic peptide substrates at a concentration of 10 μM inAssay Buffer (NEP: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% polyethyleneglycol sorbitan monolaurate (Tween-20), 10 μM ZnSO₄; ACE: 50 mM HEPES,pH 7.5, 100 mM NaCl, 0.01% Tween-20, 1 μM ZnSO₄). The respective enzymeswere used at concentrations that resulted in quantitative proteolysis of1 μM of substrate after 20 minutes at 37° C.

Test compounds were assayed over the range of concentrations from 10 μMto 20 pM. Test compounds were added to the enzymes and incubated for 30minute at 37° C. prior to initiating the reaction by the addition ofsubstrate. Reactions were terminated after 20 minutes of incubation at37° C. by the addition of glacial acetic acid to a final concentrationof 3.6% (v/v).

Plates were read on a fluorometer with excitation and emissionwavelengths set to 320 nm and 405 nm, respectively. Inhibition constantswere obtained by nonlinear regression of the data using the equation(GraphPad Software, Inc., San Diego, Calif.):v=v ₀/[1+(I/K′)]where v is the reaction rate, v₀ is the uninhibited reaction rate, I isthe inhibitor concentration and K′ is the apparent inhibition constant.

The compound of formula I′ (Example 1A) was tested in this assay andfound to have a pK_(i) value at human NEP of ≧9.0, and the followingprodrugs were also found to have activity:

Ex. pK_(i) 1G ≧9.0 1I 7.0-7.9 1L ≧9.0 1M 8.0-8.9The prodrug compounds of Examples 1B-F, 1H, 1J-K and 1N-P were nottested since activity would not be expected in this in vitro assay;however, based upon the activity of the active form, the prodrugs areexpected to have in vivo NEP activity.

The compound of formula II′ (Example 2A) was tested in this assay andfound to have a pK_(i) value at human NEP of ≧9.0. The prodrug compoundsof Examples 2B-I either did not inhibit the enzyme in this in vitroassay, or were not tested since activity would not be expected in thisassay; however, based upon the activity of the active form, the prodrugsare expected to have in vivo NEP activity.

The compound of formula III′ (Example 3A) was tested in this assay andfound to have a pK_(i) value at human NEP of ≧9.0. The prodrug compoundsof Examples 3B-E either did not inhibit the enzyme in this in vitroassay, or were not tested since activity would not be expected in thisassay; however, based upon the activity of the active form, the prodrugsare expected to have in vivo NEP activity.

The following compounds of the invention were tested in this assay andfound to have pK_(i) values at human NEP as follows. In general, eitherthe prodrug compounds did not inhibit the enzyme in this in vitro assay,or the prodrugs were not tested (n.d.) since activity would not beexpected.

Ex. pK_(i) 4A n.d. 4B ≧9.0 5A n.d. 5B ≧9.0 6A ≧9.0 6B n.d. 6C n.d. 6Dn.d. 6E n.d.

The compound of formula VII′ (Example 7A) was tested in this assay andfound to have a pK_(i) value at human NEP of ≧9.0. The prodrug compoundsof Examples 7B-E either did not inhibit the enzyme in this in vitroassay, or were not tested since activity would not be expected in thisassay; however, based upon the activity of the active form, the prodrugsare expected to have in vivo NEP activity.

The compound of formula VIII′ (Example 8A) was tested in this assay andfound to have a pK_(i) value at human NEP of ≧9.0. The prodrug compoundsof Examples 8B-L either did not inhibit the enzyme in this in vitroassay, or were not tested since activity would not be expected in thisassay; however, based upon the activity of the active form, the prodrugsare expected to have in vivo NEP activity. The prodrug compounds ofExample 81 are expected to have in vivo NEP activity.

The compound of formula IX′ (Example 9A) was tested in this assay andfound to have a pK_(i) value at human NEP of ≧9.0. The prodrug compoundsof Examples 9B-C either did not inhibit the enzyme in this in vitroassay, or were not tested since activity would not be expected in thisassay; however, based upon the activity of the active form, the prodrugsare expected to have in vivo NEP activity.

The compound of formula X′ was tested in this assay and found to have apK_(i) value at human NEP of ≧9.0. The prodrug compound of Example 10Aeither did not inhibit the enzyme in this in vitro assay, or was nottested since activity would not be expected in this assay; however,based upon the activity of the active form, this prodrug is expected tohave in vivo NEP activity.

The prodrug compound of Example 11A either did not inhibit the enzyme inthis in vitro assay, or was not tested since activity would not beexpected in this assay; however, this prodrug is expected to have invivo NEP activity.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statutes and regulations, all publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety to the same extent as if each document hadbeen individually incorporated by reference herein.

What is claimed is:
 1. A compound of: (a)(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester; (b) (2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicacid 2,2,3,3,3-pentafluoropropyl ester; (c)(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid5-methyl-2-oxo[1,3]dioxol-4-ylmethyl ester; or (d)(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acidbutyryloxymethyl ester.
 2. A compound of: (a)(2R,4R)-4-amino-5-(5′-chloro-2′-fluoro-biphenyl-4-yl)-2-hydroxypentanoicacid; (b)(2R,4R)-4-amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxypentanoicacid ethyl ester; (c)(2R,4R)-4-amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxypentanoicacid 5-methyl-2-oxo-[1,3]dioxol-4-ylmethyl ester; (d)(2R,4R)-4-amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxypentanoicacid 2,2,3,3,3-pentafluoropropyl ester; or (e)(2R,4R)-4-amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxypentanoicacid butyryloxymethyl ester.